[0001] The present invention is related to antibodies and compositions for diagnosis and
treatment of amyloidosis, a group of disorders and abnormalities associated with amyloid
protein such as Alzheimer's disease.
[0002] Amyloidosis is not a single disease entity but rather a diverse group of progressive
disease processes characterized by extracellular tissue deposits of a waxy, starch-like
protein called amyloid, which accumulates in one or more organs or body systems. As
the amyloid deposits accumulate, they begin to interfere with the normal function
of the organ or body system. There are at least 15 different types of amyloidosis.
The major forms are primary amyloidosis without known antecedent, secondary amyloidosis
following some other condition, and hereditary amyloidosis.
[0003] Secondary amyloidosis occurs during chronic infection or inflammatory disease, such
as tuberculosis, a bacterial infection called familial Mediterranean fever, bone infections
(osteomyelitis), rheumatoid arthritis, inflammation of the small intestine (granulomatous
ileitis), Hodgkin's disease, and leprosy.
[0004] Amyloid deposits include amyloid P (pentagonal) component (AP), a glycoprotein related
to normal serum amyloid P (SAP), and sulphated glycosaminoglycans (GAG), complex carbohydrates
of connective tissue. Amyloid protein fibrils, which account for about 90% of the
amyloid material, comprise one of several different types of proteins. These proteins
are capable of folding into so-called "beta-pleated" sheet fibrils, a unique protein
configuration which exhibits binding sites for Congo red resulting in the unique staining
properties of the amyloid protein.
[0005] Many diseases of aging are based on or associated with amyloid-like proteins and
are characterized, in part, by the buildup of extracellular deposits of amyloid or
amyloid-like material that contribute to the pathogenesis, as well as the progression
of the disease. These diseases include, but are not limited to, neurological disorders
such as Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome, hereditary
cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex.
Other diseases which are based on or associated with amyloid-like proteins are progressive
supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease,
HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile
cardiac amyloidosis; endocrine tumors, and others, including macular degeneration.
[0006] Although pathogenesis of these diseases may be diverse, their characteristic deposits
often contain many shared molecular constituents. To a significant degree, this may
be attributable to the local activation of pro-inflammatory pathways thereby leading
to the concurrent deposition of activated complement components, acute phase reactants,
immune modulators, and other inflammatory mediators (McGeer et al., 1994).
[0007] Alzheimer's Disease (AD) is a neurological disorder primarily thought to be caused
by amyloid plaques, an accumulation of abnormal deposit of proteins in the brain.
The most frequent type of amyloid found in the brain of affected individuals is composed
primarily of A
β fibrils. Scientific evidence demonstrates that an increase in the production and
accumulation of beta-amyloid protein in plaques leads to nerve cell death, which contributes
to the development and progression of AD. Loss of nerve cells in strategic brain areas,
in turn, causes reduction in the neurotransmitters and impairment of memory. The proteins
principally responsible for the plaque build up include amyloid precursor protein
(APP) and two presenilins (presenilin I and presenilin II). Sequential cleavage of
the amyloid precursor protein (APP), which is constitutively expressed and catabolized
in most cells, by the enzymes
β and
γ secretase leads to the release of a 39 to 43 amino acid A
β peptide. The degradation of APPs likely increases their propensity to aggregate in
plaques. It is especially the A
β(1-42) fragment that has a high propensity of building aggregates due to two very
hydrophobic amino acid residues at its C-terminus. The A
β(1-42) fragment is therefore believed to be mainly involved and responsible for the
initiation of neuritic plaque formation in AD and to have, therefore, a high pathological
potential. There is therefore a need for agents to prevent the formation of amyloid
plaques and to diffuse existing plaques in AD.
[0008] The symptoms of AD manifest slowly and the first symptom may only be mild forgetfulness.
In this stage, individuals may forget recent events, activities, the names of familiar
people or things and may not be able to solve simple math problems. As the disease
progresses, symptoms are more easily noticed and become serious enough to cause people
with AD or their family members to seek medical help. Mid-stage symptoms of AD include
forgetting how to do simple tasks such as grooming, and problems develop with speaking,
understanding, reading, or writing. Later stage AD patients may become anxious or
aggressive, may wander away from home and ultimately need total care.
[0009] Presently, the only definite way to diagnose AD is to identify plaques and tangles
in brain tissue in an autopsy after death of the individual. Therefore, doctors can
only make a diagnosis of "possible" or "probable" AD while the person is still alive.
Using current methods, physicians can diagnose AD correctly up to 90 percent of the
time using several tools to diagnose "probable" AD. Physicians ask questions about
the person's general health, past medical problems, and the history of any difficulties
the person has carrying out daily activities. Behavioral tests of memory, problem
solving, attention, counting, and language provide information on cognitive degeneration
and medical tests such as tests of blood, urine, or spinal fluid, and brain scans
can provide some further information.
[0010] The management of AD consists of medication-based and non-medication based treatments.
Treatments aimed at changing the underlying course of the disease (delaying or reversing
the progression) have so far been largely unsuccessful. Medicines that restore the
deficit (defect), or malfunctioning, in the chemical messengers of the nerve cells
(neurotransmitters), in particular the cholinesterase inhibitors (ChEIs) such as tacrine
and rivastigmine, have been shown to improve symptoms. ChEIs impede the enzymatic
degradation of neurotransmitters thereby increasing the amount of chemical messengers
available to transmit the nerve signals in the brain.
[0011] For some people in the early and middle stages of the disease, the drugs tacrine
(COGNEX®, Morris Plains, NJ), donepezil (ARICEPT®, Tokyo, JP), rivastigmine (EXELON®,
East Hanover, NJ), or galantamine (REMINYL®, New Brunswick, NJ) may help prevent some
symptoms from becoming worse for a limited time. Another drug, memantine (NAMENDA®,
New York, NY), has been approved for treatment of moderate to severe AD. Medications
are also available to address the psychiatric manifestations of AD. Also, some medicines
may help control behavioral symptoms of AD such as sleeplessness, agitation, wandering,
anxiety, and depression. Treating these symptoms often makes patients more comfortable
and makes their care easier for caregivers. Unfortunately, despite significant treatment
advances showing that this class of agents is consistently better than a placebo,
the disease continues to progress, and the average effect on mental functioning has
only been modest. Many of the drugs used in AD medication such as, for example, ChEIs
also have side effects that include gastrointestinal dysfunction, liver toxicity and
weight loss.
[0012] Another disease that is based on or associated with the accumulation and deposit
of amyloid-like protein is macular degeneration.
[0013] Macular degeneration is a common eye disease that causes deterioration of the macula,
which is the central area of the retina (the paper-thin tissue at the back of the
eye where light-sensitive cells send visual signals to the brain). Sharp, clear, 'straight
ahead' vision is processed by the macula. Damage to the macula results in the development
of blind spots and blurred or distorted vision. Age-related macular degeneration (AMD)
is a major cause of visual impairment in the United States and for people over age
65 it is the leading cause of legal blindness among Caucasians. Approximately 1.8
million Americans age 40 and older have advanced AMD, and another 7.3 million people
with intermediate AMD are at substantial risk for vision loss. The government estimates
that by 2020 there will be 2.9 million people with advanced AMD. Victims of AMD are
often surprised and frustrated to find out how little is known about the causes and
treatment of this blinding condition.
[0014] There are two forms of macular degeneration: dry macular degeneration and wet macular
degeneration. The dry form, in which the cells of the macula slowly begin to break
down, is diagnosed in 85 percent of macular degeneration cases. Both eyes are usually
affected by dry AMD, although one eye can lose vision while the other eye remains
unaffected. Drusen, which are yellow deposits under the retina, are common early signs
of dry AMD. The risk of developing advanced dry AMD or wet AMD increases as the number
or size of the drusen increases. It is possible for dry AMD to advance and cause loss
of vision without turning into the wet form of the disease; however, it is also possible
for early-stage dry AMD to suddenly change into the wet form.
The wet form, although it only accounts for 15 percent of the cases, results in 90
percent of the blindness, and is considered advanced AMD (there is no early or intermediate
stage of wet AMD). Wet AMD is always preceded by the dry form of the disease. As the
dry form worsens, some people begin to have abnormal blood vessels growing behind
the macula. These vessels are very fragile and will leak fluid and blood (hence 'wet'
macular degeneration), causing rapid damage to the macula.
[0015] The dry form of AMD will initially often cause slightly blurred vision. The center
of vision in particular may then become blurred and this region grows larger as the
disease progresses. No symptoms may be noticed if only one eye is affected. In wet
AMD, straight lines may appear wavy and central vision loss can occur rapidly.
[0016] Diagnosis of macular degeneration typically involves a dilated eye exam, visual acuity
test, and a viewing of the back of the eye using a procedure called fundoscopy to
help diagnose AMD, and-if wet AMD is suspected-fluorescein angiography may also be
performed. If dry AMD reaches the advanced stages, there is no current treatment to
prevent vision loss. However, a specific high dose formula of antioxidants and zinc
may delay or prevent intermediate AMD from progressing to the advanced stage. Macugen®
(pegaptanib sodium injection), laser photocoagulation and photodynamic therapy can
control the abnormal blood vessel growth and bleeding in the macula, which is helpful
for some people who have wet AMD; however, vision that is already lost will not be
restored by these techniques. If vision is already lost, low vision aids exist that
can help improve the quality of life.
[0017] One of the earliest signs of age-related macular degeneration (AMD) is the accumulation
of extracellular deposits known as drusen between the basal lamina of the retinal
pigmented epithelium (RPE) and Bruch's membrane (BM). Recent studies conducted by
Anderson et al. have confirmed that drusen contains amyloid beta. (
Experimental Eye Research 78 (2004) 243-256).
[0018] Ongoing research continues with studies exploring environmental, genetic, and dietary
factors that may contribute to AMD. New treatment strategies are also being explored,
including retinal cell transplants, drugs that will prevent or slow down the progress
of the disease, radiation therapy, gene therapies, a computer chip implanted in the
retina that may help stimulate vision and agents that will prevent the growth of new
blood vessels under the macula.
[0019] An important factor to consider when developing new drugs is the ease of use for
the target patients. Oral drug delivery, -specifically tablets, capsules and softgels-,
account for 70% of all dosage forms consumed because of patient convenience. Drug
developers agree that patients prefer oral delivery rather than subjecting themselves
to injections or other, more invasive forms of medicinal administration. Formulations
resulting in low dosing intervals (i.e. once a day or sustained release) are also
preferable. The ease of administering antibiotics in oral dosage forms results in
an increase of patient compliance during treatment.
[0020] What is needed are effective methods and compositions for preventing or addressing
the complications associated with amyloidosis, a group of diseases and disorders associated
with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis
including, but not limited to, neurological disorders such as Alzheimer's Disease
(AD), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis
(Dutch type); the Guam Parkinson-Dementia complex; as well as other diseases which
are based on or associated with amyloid-like proteins such as progressive supranuclear
palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related
dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac
amyloidosis; endocrine tumors, and others, including macular degeneration. In particular
what is needed are agents capable of counteracting the physiological manifestations
of the disease such as the formation of plaques associated with aggregation of fibers
of the amyloid or amyloid-like peptide.
[0021] Anti-amyloid antibodies elicited by the inoculation of A
β1-42 mixed with Freund complete or incomplete adjuvant were reported to reduce the amyloid
burden in transgenic mice for human Alzheimer disease (Schenk et al., 1999). Intraperitoneal
inoculation of tetrapalmitoylated A
β1-16 reconstituted in liposomes to NORBA transgenic mice elicited significant titers of
anti-amyloid antibodies, which were reported to solubilize amyloid fibers and plaques
in vitro and
in vivo. (Nicolau et al., 2002).
[0022] WO 01/62801 A2 discloses an anti-β-amyloid humanized antibody of the 266 antibody and suggests that
antibodies that bind β-amyloid between positions 13 and 28 (
e.g., 266 and 4G8) are capable of sequestering soluble forms of β-amyloid from their bound,
circulating forms in the blood without binding with great affinity to aggregated β-amyloid.
[0023] WO 03/070760 A2 discloses an antibody molecule that recognizes a conformational epitope on the β-A4
peptide, which is defined by the amino acid sequences AEFRHDSGY and VHHQKLVFFAEDVG.
[0024] WO 2006/066171 A1 discloses an anti-13-amyloid antibody (15C11) that binds to the central region of
β-amyloid (
i.e., amino acids 19-22) and binds oligomeric β-amyloid species, but does not appear to
bind to β-amyloid monomers.
[0025] A possible mechanism by which the dissolution of amyloid plaques and fibres occurred
was first suggested by Bard et al., (2000), who concluded that the antibodies opsonized
the plaques, which were subsequently destroyed by the macrophages of the microglia.
De Mattos et al., (2001) indicated that a mAb directed against the central domain
of
β-amyloid was able to bind and completely sequester plasma amyloid. They argued that
the presence of these mAbs in circulation shifted the equilibrium of A
β between brain and plasma, favoring the peripheral clearing and catabolism instead
of deposition within the brain.
[0026] Prolonged human therapy with rodent antibodies may result in an antiglobulin response
which is detectable at about 8-12 days after administration and reaches a peak at
about 20-30 days. If such an antiglobulin response is encountered, the treatment must
be discontinued after not more than about 10 days and re-treatment at a latter date
is usually precluded because it will lead to rapid onset of a secondary antiglobulin
response. Although rodent antibodies share a considerable degree of sequence conservation
with that of human antibodies, there are many sequence differences between rodents
and human antibodies sufficient for the rodent antibodies to be immunogenic in humans.
[0027] This problem may be overcome by generating antibodies directly in humans or by the
creation of "humanized' (a.k.a. "reshaped' antibodies). Humanized antibodies have
a variable region amino acid sequence that contains the rodent-derived CDRs interspersed
into human or human-like framework sequences. Since the specificity of the humanized
antibody is provided by the rodent-derived CDRs, their residues are to be used essentially
unchanged with only minor modifications being allowable, which do not significantly
interfere with the affinity and specificity of the antibody for its target antigen.
Framework residues may be derived from any primate or, particularly, from any human
variable region or may be a combination thereof and the resultant designed variable
region would be considered reshaped.
[0028] To maximise the likelihood that affinity will be retained in the reshaped antibody
it is important to make a proper selection of the framework region. It is known that
the framework sequences serve to hold the CDRs in their correct spatial orientation
for interaction with antigen, and that framework residues can sometimes even participate
in antigen binding. In order to maintain the affinity of the antibody for its antigen
it is advantageous to select human framework sequences that are most similar to the
sequences of the rodent frameworks. It then may still be necessary to replace one
or more amino acids in the human framework sequence with the corresponding residue
in the rodent framework to avoid losses with the affinity. This replacement may be
aided by computer modelling.
[0029] The present invention provides novel antibodies and compositions comprising highly
specific and highly effective antibodies, namely humanized antibodies including fragments
thereof, having the ability to specifically recognize and bind to specific epitopes
from a range of
β-amyloid antigens, which my be presented to the antibody in a monomeric, dimeric,
trimeric, etc, a polymeric form, in form of an aggregate, fibers, filaments or in
the condensed form of a plaque. The antibodies enabled by the teaching of the present
invention are particularly useful for the treatment of amyloidosis, a group of diseases
and disorders associated with amyloid plaque formation including secondary amyloidosis
and age-related amyloidosis including, but not limited to, neurological disorders
such as Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome, hereditary
cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex;
as well as other diseases which are based on or associated with amyloid-like proteins
such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,
hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, HIV-related
dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac
amyloidosis; endocrine tumors, and others, including macular degeneration, to name
just a few.
[0030] Surprisingly, the antibodies of the invention bind to β-amyloid in various forms
of β-amyloid, including soluble monomers, oligomers and fibrils, with high affinity,
and they inhibit aggregation of monomers into oligomers/fibrils and induce disaggregation
of oligomers/fibrils.
[0031] The invention provides a humanized antibody or a fragment thereof, wherein said antibody
or fragment thereof comprises:
- (A) (i) a Heavy Chain Variable Region (HCVR) having an amino acid sequence that is
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15,
wherein the HCVR comprises a heavy chain complementarity determining region 1 (CDR1)
as given in SEQ ID NO: 1, a heavy chain CDR2 as given in SEQ ID NO: 2 and a heavy
chain CDR3 as given in SEQ ID NO: 3, and (ii) a light chain variable region (LCVR)
comprising a light chain CDR1 as given in SEQ ID NO: 4, a light chain CDR2 as given
in SEQ ID NO: 5, RVSNRFS or KVSSRFS and a light chain CDR3 as given in SEQ ID NO:
6, or
- (B) (i) an HCVR comprising a heavy chain CDR1 as given in SEQ ID NO: 1, a heavy chain
CDR2 as given in SEQ ID NO: 2 and a heavy chain CDR3 as given in SEQ ID NO: 3, and
(ii) a LCVR having an amino acid sequence that is 96%, 97%, 98%, 99%, or 100% identical
to the amino acid sequence of SEQ ID NO: 12, wherein the LCVR comprises a light chain
CDR1 as given in SEQ ID NO: 4, a light chain CDR2 as given in SEQ ID NO: 5, RVSNRFS
or KVSSRFS and a light chain CDR3 as given in SEQ ID NO: 6,
wherein said antibody or fragment thereof specifically binds to
β-amyloid protein.
[0032] In some embodiments, the humanized antibody of the invention or fragment thereof
as defined in 1(A) above, comprises the LCVR having an amino acid sequence that is
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12.
[0033] In some embodiments, the humanized antibody of the invention or fragment thereof
as defined in 1(B) above, comprises the HCVR having an amino acid sequence that is
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15.
[0034] In some embodiments, the humanized antibody of the invention or a fragment thereof
comprises:
- i) the HCVR having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence
that is 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
15; or
- ii) the LCVR having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence
that is 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
12; or
- iii) the HCVR having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence
that is 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
15 and the LCVR having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence
that is 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
12.
[0035] In some embodiments, the humanized antibody of the invention or fragment thereof
comprises:
- i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid
sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the amino acid sequence of SEQ ID NO: 16; or
- ii) a light chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid
sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the amino acid sequence of SEQ ID NO: 13; or
- iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 or an amino
acid sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino
acid sequence of SEQ ID NO: 13 or an amino acid sequence that is 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
13.
[0036] In some embodiments, the humanized antibody of the invention or fragment thereof
is of the IgG4 isotype.
[0037] In some embodiments, the humanized antibody of the invention or fragment thereof
is capable of inhibiting the aggregation of
β-amyloid monomers to high molecular polymeric fibrils and of disaggregating preformed
polymeric fibrils or filaments.
[0038] In some embodiments, the humanized antibody of the invention or fragment thereof
binds to an Aβ fiber, fibril or filament with a binding affinity that is at least
10 times, at least 15 times, at least 20 times, or at least 25 times, higher than
the binding affinity of said antibody or fragment thereof to an Aβ monomer.
[0039] In some embodiments, the humanized antibody of the invention or fragment thereof
has:
- (a) the amino acid in Kabat position 47 of the framework regions of the Heavy Chain
Variable Region is the amino acid Leu; and/or
- (b) the amino acid in Kabat position 94 in the framework regions of the Heavy Chain
Variable Region is the amino acid Ser; and/or
- (c) the amino acid in Kabat position 87 in the framework regions of the Light Chain
Variable Region is the amino acid Tyr, Phe, Leu, VaI, Ile, or Ala.
[0040] The invention further provides a nucleic acid molecule comprising a nucleotide sequence
encoding the humanized antibody of the invention or fragment thereof.
[0041] The invention further provides an expression vector comprising the nucleic acid molecule
of the invention.
[0042] The invention further provides a cell comprising the expression vector of the invention.
[0043] The invention further provides a composition comprising the humanized antibody of
the invention or fragment thereof, and further comprising a pharmaceutically acceptable
carrier, a diluent, and/or an excipient.
[0044] The invention further provides a mixture comprising the humanized antibody of the
invention or fragment thereof, and further comprising a biologically active substance,
a pharmaceutically acceptable carrier, a diluent, and/or an excipient.
[0045] In some embodiments, the mixture comprises:
- (i) a biologically active substance that is a compound used in the treatment of amyloidosis,
or
- (ii) at least one of the following compounds: an anti-oxidative stress compound; an
anti-apoptotic compound; a metal chelator; a DNA repair inhibitor such as pirenzepine
and metabolites; 3-amino-1-propanesulfonic acid (3 APS); 1,3-propanedisulfonate (1,3PDS);
α-secretase activator; Aβ-secretase inhibitor; a γ-secretase inhibitor; a tau protein;
a neurotransmitter; aβ-sheet breaker; an attractant for amyloid beta clearing / depleting
cellular components; an inhibitor of N-terminal truncated amyloid beta, such as pyroglutamated
amyloid beta 3-42; an anti-inflammatory molecule; a cholinesterase inhibitor (ChEI)
such as tacrine, rivastigmine, donepezil, and/or galantamine; an Ml agonist; or another
drug such as an amyloid- or tau-modifying drug or nutritive supplement.
[0046] The invention further provides a humanized antibody or fragment thereof as defined
in any one of claims 1-9, a composition as defined in claim 13, or a mixture as defined
in claim 14 or 15 for use in a method of treating or alleviating the effects of amyloidosis
in an animal, such as a mammal or a human.
[0047] In some embodiments, the amyloidosis is secondary amyloidosis, age-related amyloidosis,
a neurological disorder, Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome,
hereditary cerebral hemorrhage with amyloidosis (Dutch type), the Guam Parkinson-Dementia
complex, progressive supranuclear palsy, multiple sclerosis, Creutzfeld Jacob disease,
Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult
Onset Diabetes, senile cardiac amyloidosis, endocrine tumors, or macular degeneration.
[0048] In some embodiments, the amyloidosis is Alzheimer's disease.
[0049] In some embodiments, the treatment of the animal according to the invention leads
to
- i) an increase of the cognitive memory capacity and/or;
- ii) the retention of cognitive memory capacity; and/or
- iii) a complete restoration of the cognitive memory capacity.
[0050] The invention further provides a method of diagnosis of an amyloidosis in a patient
comprising:
- i) contacting a sample, body part, or body area containing an amyloid protein with
the humanized antibody of the invention or fragment thereof under conditions that
allow binding of said antibody or fragment thereof to the amyloid protein; and
- ii) detecting the antibody or fragment thereof bound to the protein,
wherein the presence or absence of antibody or fragment thereof bound to the amyloid
protein indicates the presence or absence of amyloid protein in said sample, body
part, or body area.
[0051] The invention further provides a method of determining the extent of amyloidogenic
plaque burden in a tissue sample or body fluid sample comprising:
- i) testing a tissue sample or body fluid sample for the presence of amyloid protein
with the humanized antibody of the invention or fragment thereof;
- ii) determining the amount of antibody or fragment thereof bound to the amyloid protein;
and
- iii) calculating the plaque burden in the tissue sample or body fluid sample.
[0052] The invention further provides a kit for detection and diagnosis of amyloid-associated
diseases and conditions comprising the humanized antibody of the invention or fragment
thereof, wherein said kit comprises a container holding one or more antibodies of
the invention or fragments thereof and instructions for using said antibodies or fragments
thereof.
[0053] The invention further provides a humanized antibody of the invention or fragment
thereof, a composition of the invention, or a mixture of the invention, for use in
a method of preventing degeneration of neurons upon exposure to β-amyloid oligomers.
[0054] The invention further provides a humanized antibody of the invention or fragment
thereof, a composition of the invention, or a mixture of the invention, for use in
a method of protecting neurons from β-amyloid-induced degradation.
[0055] The invention further provides a method for preparing a humanized antibody of the
invention or fragment thereof, wherein the method comprises culturing the cell of
the invention, and purifying the antibody or fragment thereof.
SUMMARY OF THE INVENTION
[0056] In one embodiment, the invention relates to a humanized antibody or a fragment thereof,
which recognizes and binds to at least one distinct binding site, particularly to
a least two distinct binding sites, and more particularly to at least three distinct
binding sites on the
β-amyloid protein wherein said one, said at least two and said at least three binding
sites each comprise at least one or two consecutive amino acid residues predominantly
involved in the binding of the antibody.
[0057] In particular, the humanized antibody or a fragment thereof described therein binds
to at least two, particularly to at least three distinct binding sites on the
β-amyloid protein wherein at least two of the three distinct binding sites comprise
at least two consecutive amino acid residues predominantly involved in the binding
of the antibody and at least one of the three distinct binding sites comprise at least
one amino acid residue.
[0058] The at least two distinct binding sites comprising at least two consecutive amino
acid residues predominantly involved in the binding of the antibody are located in
close proximity to each other on the antigen, separated and/or flanked by at least
one amino acid residue not involved in antibody binding or to a significantly smaller
extent as compared to said at least two consecutive amino acid residues, thus forming
a conformational discontinuous epitope.
[0059] The at least three distinct binding sites comprising at least two consecutive amino
acid residues and at least one amino acid residue, respectively, which are predominantly
involved in the binding of the antibody are located in close proximity to each other
on the epitope, separated and/or flanked by at least one amino acid residue not involved
in antibody binding or to a significantly smaller extent as compared to the amino
acid residues, which are predominantly involved in the binding of the antibody, thus
forming a conformational discontinuous epitope.
[0060] In particular, a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof is disclosed, which recognizes and binds to at least one distinct
binding site, particularly to a least two distinct binding sites, more particularly
to at least three distinct binding sites on the
β-amyloid- protein wherein said at least one or said at least two distinct binding
sites each comprise at least two consecutive amino acid residues predominantly involved
in the binding of the antibody, wherein the at least two consecutive amino acid residues
representing a first binding site are -Phe-Phe- embedded within the following core
sequence (SEQ ID NO: 9):
Xaa3 - Phe - Phe - Xaa4 - Xaa5 - Xaa6, wherein
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile;
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp,
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, and wherein
said amino acid residues Xaa3 Xaa4, Xaa5 and Xaa6 are not involved in antibody binding or to a significantly smaller extent as compared
to the -Phe-Phe- binding site.
[0061] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, wherein
Xaa3 is Val or Leu, but particularly Val;
Xaa4 is Ala or Val, but particularly Ala;
Xaa5 is Glu or Asp, but particularly Glu;
Xaa6 is Glu or Asp, but particularly Asp.
[0062] In particular, a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof is disclosed, which recognizes and binds to at least one distinct
binding site, particularly to a least two distinct binding sites, more particularly
to at least three distinct binding sites on the
β-amyloid protein wherein said distinct binding sites comprise at least one and at
least two consecutive amino acid residues, respectively, predominantly involved in
the binding of the antibody, wherein the at least two consecutive amino acid residues
representing a first binding site are -Phe-Phe- and the at least one amino acid residue
is -His- embedded within the following core sequence:
- Xaa1 - His - Xaa3 - Xaa4 - Xaa5 - Xaa6 - Phe - Phe - Xaa7 -Xaa8- Xaa9-, wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys
and Arg
Xaa3 is an amino acid residue selected from the group consisting of Asn and Gln
Xaa4 is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys
and Arg
Xaa5 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile;
Xaa6 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile
Xaa7 is an amino acid residue selected from the group consisting of Ala, Val, Leu and
Ile Xaa8 is an amino acid residue selected from the group consisting of Glu and Asp,
Xaa9 is an amino acid residue selected from the group consisting of Glu and Asp, and wherein
said amino acid residues Xaa1, Xaa3, Xaa6, Xaa7, Xaa8 and Xaa9, are not involved in antibody binding or to a smaller to significantly smaller extent
as compared to the -His- and the -Phe-Phe- binding site, respectively..
[0063] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, wherein
Xaa3 is Gln or Asn, but particularly Gln;
Xaa4 is Lys
Xaa5 is Leu
Xaa6 is Val or Leu, but particularly Val;
Xaa7 is Ala or Val, but particularly Ala;
Xaa8 is Glu or Asp, but particularly Glu; and
Xaa9 is Asp or Glu, but particularly Asp.
[0064] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, which recognizes and binds to at least one distinct binding
site, particularly to a least two distinct binding sites, more particularly to at
least three distinct binding sites on the
β-amyloid protein, wherein said at least one or said at least two distinct binding
sites each comprise at least two consecutive amino acid residues predominantly involved
in the binding of the antibody, wherein the at least two consecutive amino acid residues
representing a second binding site are -Lys-Leu- embedded within the following core
sequence (SEQ ID NO: 10):
Xaa1 - Xaa2 - Lys - Leu - Xaa3 wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln Lys,
and Arg;
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile; and wherein said amino acid residues Xaa2, Xaa3, are not involved in antibody binding or to a smaller to significantly smaller extent
as compared to the -Lys-Leu- binding site.
[0065] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, which recognizes and binds to at least one distinct binding
site, particularly to a least two distinct binding sites, more particularly to at
least three distinct binding sites on the
β-amyloid protein wherein said distinct binding sites comprise at least one and at
least two consecutive amino acid residues, respectively, predominantly involved in
the binding of the antibody, wherein the at least one and the at least two consecutive
amino acids, which are separated by at least one amino acid residue not involved in
antibody binding or to a significantly smaller extent as compared to the amino acid
residues predominantly involved in the binding of the antibody, are -His- and -Lys-Leu-,
respectively, embedded within the following core sequence:
His - Xaa2 - Lys - Leu - Xaa3- Xaa4- Xaa5-Xaa6- - Xaa7 - Xaa8 - wherein
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile;
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile
Xaa5 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile
Xaa6 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile;
Xaa7 is an amino acid residue selected from the group consisting of Glu and Asp,
Xaa8 is an amino acid residue selected from the group consisting of Glu and Asp
and wherein said amino acid residues Xaa2, Xaa3, Xaa6, Xaa7, Xaa8, are not involved in antibody binding or to a smaller to significantly smaller extent
as compared to the -His- and the -Lys-Leu- binding site, respectively.
[0066] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, wherein
Xaa2 is Gln or Asn, but particularly Gln;
Xaa3 is Val or Leu, but particularly Val;
Xaa4 is Phe
Xaa5 is Phe
Xaa6 is Ala or Val, but particularly Ala;
Xaa7 is Glu or Asp, but particularly Glu; and
Xaa8 is Asp or Glu, but particularly Asp.
[0067] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, which recognizes and binds to at least two distinct binding
sites on the
β-amyloid protein wherein said at least two distinct binding sites each comprise at
least two consecutive amino acid residues predominantly involved in the binding of
the antibody, wherein the at least two consecutive amino acids are separated by at
least one amino acid residue not involved in antibody binding or to a significantly
smaller extent than said consecutive amino acid residues, which are -Phe-Phe- and
-Lys-Leu-, respectively, representing a first and second binding site embedded within
the following core sequence:
Xaa1 - Xaa2 - Lys - Leu - Xaa3 - Phe - Phe - Xaa4 - Xaa5 - Xaa6, wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln Lys,
and Arg;
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile;
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp,
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp and wherein
said amino acid residues Xaa2, Xaa3, Xaa4, Xaa5 and Xaa6 are not involved in antibody binding or to a smaller to significantly smaller extent
as compared to the -Lys-Leu- and - Phe-Phe- binding site, respectively.
[0068] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, which recognizes and binds to at least one distinct binding
site, particularly to a least two distinct binding sites, more particularly to at
least three distinct binding sites on the
β-amyloid protein wherein said distinct binding sites comprise at least one and at
least two consecutive amino acid residues, respectively, predominantly involved in
the binding of the antibody, wherein the at least one and the at least two consecutive
amino acids are separated by at least one amino acid residue not involved in antibody
binding or to a significantly smaller extent as compared to the amino acid residues,
which are predominantly involved in the binding of the antibody, and wherein said
amino acid residues are -His- and -Phe-Phe- and -Lys-Leu-, respectively, embedded
within the following core sequence:
His - Xaa2 - Lys - Leu - Xaa3 - Phe - Phe - Xaa4 - Xaa5 - Xaa6, wherein
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile;
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp,
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, and wherein
said amino acid residues Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, are not involved in antibody binding or to a smaller to significantly smaller extent
as compared to the -His-, the -Lys-Leu- and the -Phe-Phe- binding site, respectively.
[0069] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, wherein
Xaa2 is Gln or Asn, but particularly Gln;
Xaa3 is Val or Leu, but particularly Val;
Xaa4 is Ala or Val, but particularly Ala;
Xaa5 is Glu or Asp, but particularly Glu; and
Xaa6 is Asp or Glu, but particularly Asp.
[0070] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, which recognizes and binds to at least two distinct binding
sites on the
β-amyloid protein wherein said at least two distinct binding sites each comprise at
least two consecutive amino acid residues predominantly involved in the binding of
the antibody, wherein the at least two consecutive amino acids are separated by at
least one amino acid residue not involved in antibody binding or to a significantly
smaller extent than said consecutive amino acid residues, which are -Phe-Phe- and
-Lys-Leu-, respectively, representing a first and second binding site embedded within
the following core sequence:
Xaa1 - Xaa2 - Lys - Leu - Xaa3 - Phe - Phe - Xaa4 - Xaa5 - Xaa6, wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys
and Arg;
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln;
Xaa3 is an amino acid residue selected from the group consisting of Val, Ala, Leu, Met,
Phe, norleucine and Ile
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu and
Ile;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp,
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, and wherein
said amino acid residues Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, are not involved in antibody binding or to a smaller to significantly smaller extent
as compared to the -Lys-Leu- and the -Phe- Phe binding site, respectively.
[0071] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, wherein
Xaa1 is His or Arg, but particularly His;
Xaa2 is Gln or Asn, but particularly Gln;
Xaa3 is Val or Leu, but particularly Val;
Xaa4 is Ala or Val, but particularly Ala;
Xaa5 is Glu or Asp, but particularly Glu; and
Xaa6 is Asp or Glu, but particularly Asp.
[0072] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof which recognizes and binds to at least two distinct binding
sites on the
β-amyloid protein wherein said at least two distinct binding sites each comprise at
least two consecutive amino acid residues predominantly involved in the binding of
the antibody, which are - Phe - Phe - Ala - Glu -, particularly - Phe - Phe - Ala
-,but especially - Phe - Phe - and - Lys - Leu -, respectively, and wherein said at
least two distinct binding sites exhibit amino acid sequence -Val - Phe - Phe - Ala
- Glu - Asp - shown in SEQ ID NO: 7 and amino acid sequence His - Gln - Lys - Leu
- Val - shown in SEQ ID NO: 8, respectively.
[0073] Also disclosed is a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof, which recognizes and binds to at least one distinct binding
site, particularly to a least two distinct binding sites, more particularly to at
least three distinct binding sites on the
β-amyloid protein wherein the said at least one or said at least two distinct binding
sites comprise at least one and at least two consecutive amino acid residues, respectively,
predominantly involved in the binding of the antibody, which are - Phe - Phe - and
- Lys - Leu -, and -His-, respectively, wherein said distinct binding sites are embedded
in the amino acid sequence -Val - Phe - Phe - Ala - Glu-, and amino acid sequence
-His - Gln - Lys - Leu - Val -, respectively.
[0074] In another aspect the chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof comprises an antigen recognition and binding site which recognizes
and binds to at least two distinct binding sites on the
β-amyloid protein wherein said at least two distinct binding sites each comprise at
least two consecutive amino acid residues within the amino acid sequence given in
SEQ ID NOs: 7 and 8, respectively, wherein said consecutive amino acid residues, particularly
-Phe- Phe- and -Lys-Leu-, are predominantly involved in the binding of the
β-amyloid protein.
[0075] Also disclosed is an antibody or a fragment thereof which binds to 4 distinct binding
sites on the
β-amyloid protein wherein said 4 distinct binding sites include 2 binding sites each
comprising one amino acid residue and 2 binding sites each comprising two consecutive
amino acid residues, which residues are predominantly involved in the binding of the
antibody, wherein said 4 distinct binding sites are located in close proximity to
each other on the
β-amyloid protein, and wherein said 4 binding sites are separated by at least one amino
acid residue not involved in antibody binding or involved in binding but to a significantly
smaller extent as compared to said one amino acid residue and said two consecutive
amino acid residues of the 4 distinct binding sites thus forming a conformational
discontinuous epitope.
[0076] In particular, the first of the two consecutive amino acid residues predominantly
involved in the binding of the antibody is -Lys-Leu-, and the second of the at least
two consecutive amino acid residues is -Phe-Phe-, the first of the single amino acid
residues is -His- and the second of the single amino acid residues is -Asp- embedded
within the following core sequence:
- Xaa1- His - Xaa2 - Lys - Leu -Xaa3 - Phe - Phe - Xaa4-Xaa5- Asp. - Xaa6 wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys
and Arg, but particularly His;
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln, but particularly
Gln ;
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile, particularly Val;
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile, particularly Ala;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp, particularly
Glu;
Xaa6 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile, particularly Val; and wherein said amino acid residues Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, are not involved in antibody binding or are involved in binding but to a significantly
smaller extent as compared to the -His-, - Asp-, the -Lys-Leu, and the -Phe-Phe- binding
site.
[0077] Also disclosed is an antibody or a fragment thereof, which binds to 4 distinct binding
sites on the
β-amyloid protein, wherein said 4 distinct binding sites include two binding sites
each comprising one amino acid residue and two binding sites each comprising two consecutive
amino acid residues, wherein the first of the two consecutive amino acid residues
predominantly involved in the binding of the antibody is -Lys-Leu-, and the second
of the at least two consecutive amino acid residues is -Phe-Phe-, the first of the
single amino acid residues is -His- and the second of the single amino acid residues
is -Asp- embedded within the following core sequence:
- Xaa1- His - Xaa2 - Lys - Leu -Xaa3 - Phe - Phe - Xaa4- Xaa5- Asp. - Xaa6 wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln, Lys
and Arg, but particularly His;
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln, but particularly
Gln ;
Xaa3 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile, particularly Val;
Xaa4 is an amino acid residue selected from the group consisting of Ala, Val, Leu, Ser
and Ile, particularly Ala;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp, particularly
Glu;
Xaa6 is an amino acid residue selected from the group consisting of Ala, Val, Leu, norleucine,
Met, Phe, and Ile, particularly Val; and wherein said amino acid residues Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, are not involved in antibody binding or are involved in binding but to a significantly
smaller extent as compared to the -His-, - Asp-, the -Lys-Leu, and the -Phe-Phe- binding
site.
[0078] In a specific aspect the recognition and binding sites as defined herein before are
forming a conformational discontinuous epitope localized in a region of the
β-amyloid protein between amino acid residue 12 to 24, particularly between residues
14 to 23, more particularly between amino acid residues 14 and 20, wherein the at
least two distinct recognition and binding sites each comprising at least 2 amino
acid residues, are located at position 16 and 17 and at position 19 and 20, respectively,
and wherein the at least one distinct recognition and binding site comprising at least
1 amino acid residue is located at position 14, which residues are predominantly involved
in the binding of the
β-amyloid protein and wherein said distinct recognition and binding sites are at least
on one side flanked by amino acid residues, particularly residues 21 and 22, and separated
by one amino acid residue located at position 15 and 18, which amino acid residues
are not directly involved in the binding of the antigen or, at least, to a substantially
smaller extent.
[0079] In still another aspect the said at least three distinct recognition and binding
sites are flanked on both sides by amino acid residues, particularly residues 12 and
13, and residues 21 and 22 and are separated by one amino acid residue located at
position 15 and 18, which amino acid residues are not directly involved in the binding
of the antigen or, at least, to a substantially smaller extent.
[0080] In a specific aspect, said consecutive amino acid residues, particularly -Lys-Leu-
at position 16 and 17 and -Phe- Phe- at position 19 and 20, which are predominantly
involved in the binding of the
β-amyloid protein, are embedded into the following core region:
Val- |
His- |
His- |
Gln- |
Lys- |
Leu- |
Val- |
Phe- |
Phe- |
Ala- |
Glu- |
Asp |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
21 |
22 |
23 |
[0081] In another specific aspect, said amino acid residues, particularly -Lys-Leu- at position
16 and 17 and -Phe- Phe- at position 19 and 20, and -His- at position 14, which are
predominantly involved in the binding of the
β-amyloid protein, are embedded into the following core region:
Val- |
His- |
His |
Gln- |
Lys- |
Leu- |
Val- |
Phe- |
Phe- |
Ala- |
Glu- |
Asp- |
Val- |
Gly- |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
21 |
22 |
23 |
24 |
25 |
[0082] In another aspect a humanized antibody or a fragment thereof is disclosed which comprises
in the light chain and heavy chain variable region, respectively, at least one CDR
of non-human origin, particularly two CDRs of non-human origin, more particularly
three CDR of non-human origin, embedded in one or more human- or primate-derived framework
regions and, optionally, a constant region derived from a human or primate source
antibody, which humanized antibody or fragment thereof is capable of specifically
recognizing and binding
β-amyloid protein, particularly a
β-amyloid monomeric peptide, more particularly a
β-amyloid polymeric peptide, even more particularly
β-amyloid fibers, fibrils or filaments in isolation or as part of a
β-amyloid plaque, at an epitope comprising the following amino acid sequence (SEQ ID
NO: 11):
Xaa1 - Xaa2 - Lys - Leu - Xaa3 - Phe - Phe- Xaa4 - Xaa5 - Xaa6, wherein
Xaa1 is an amino acid residue selected from the group consisting of His, Asn, Gln, but
particularly His;
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln, but particularly
Gln; and
Xaa3 is an amino acid residue selected from the group consisting of Val, Leu, and Ile,
but particularly Val;
Xaa4 is an amino acid residue selected from the group consisting of Ala and Val, but particularly
Ala;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp, but particularly
Glu;
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, but particularly
Asp.
[0083] In still another aspect, a humanized antibody or a fragment thereof is disclosed
which comprises in the light chain and heavy chain variable region, respectively,
at least one CDR of non-human origin, particularly two CDRs of non-human origin, more
particularly three CDR of non-human origin, embedded in one or more human- or primate-derived
framework regions and, optionally, a constant region derived from a human or primate
source antibody, which humanized antibody or fragment thereof is capable of specifically
recognizing and binding
β-amyloid protein, particularly a
β-amyloid monomeric peptide, more particularly a
β-amyloid polymeric peptide, even more particularly
β-amyloid fibers, fibrils or filaments in isolation or as part of a
β-amyloid plaque, at an epitope comprising the following amino acid sequence:
His - Xaa2 - Lys - Leu - Xaa3 - Phe - Phe- Xaa4 Xaa5 - Xaa6, wherein
Xaa2 is an amino acid residue selected from the group consisting of Asn and Gln, but particularly
Gln; and
Xaa3 is an amino acid residue selected from the group consisting of Val, Leu, and Ile,
but particularly Val;
Xaa4 is an amino acid residue selected from the group consisting of Ala and Val, but particularly
Ala;
Xaa5 is an amino acid residue selected from the group consisting of Glu and Asp, but particularly
Glu;
Xaa6 is an amino acid residue selected from the group consisting of Glu and Asp, but particularly
Glu; and wherein said amino acid residues Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, are not involved in antibody binding or to a smaller extent as compared to the -His-
and the -Lys-Leu- and the -Phe-Phe- binding site.
[0084] In a specific aspect the CDR of non-human origin is obtained from a donor antibody,
but particularly from a murine donor antibody, raised against an antigen fragment
which does not contain said distinct binding site. This shift in the epitopic region
may have at least partially been caused by the use of a supramolecular antigenic construct
comprising an antigenic peptide corresponding to the amino acid sequence of the
β-amyloid peptide, particularly of
β-amyloid peptide A
β1-16, modified with a hydrophilic moiety such as, for example, polyethylene glycol (PEG),
wherein said hydrophilic moiety is covalently bound to each of the termini of the
antigenic peptide through at least one, particularly one or two amino acids such as,
for example, lysine, glutamic acid and cysteine or any other suitable amino acid or
amino acid analogue capable of serving as a connecting device for coupling the hydrophilic
moiety to the peptide fragment, as described herein below in the immunization process.
When a PEG is used as the hydrophilic moiety, the free PEG termini are covalently
bound to phosphatidylethanolamine or any other compound suitable to function as the
anchoring element, for example, to embed the antigenic construct in the bilayer of
a liposome as described herein.
[0085] In particular, the CDR of non-human origin is obtained from a murine donor antibody
which exhibits the characteristic properties of ACI-01-Ab7C2 (also named "mC2" throughout
the application) deposited 01 December 2005 with the "Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH (DSMZ) in Braunschweig, Mascheroder Weg 1 B, 38124 Branuschweig,
under the provisions of the Budapest Treaty under accession no DSM ACC2750).
[0086] In one embodiment of the invention, the CDR of non-human origin is obtained from
murine donor antibody ACI-01-Ab7C2 (also named "mC2" throughout the application) deposited
01 December 2005 with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH (DSMZ) in Braunschweig, Mascheroder Weg 1 B, 38124 Branuschweig, under the provisions
of the Budapest Treaty under accession no DSM ACC2750).
[0087] Also the use of lipid A as part of the immunization protocol may have contributed
to a shift in the epitopic region.
[0088] Also disclosed is a humanized antibody or a fragment thereof comprising integrated
into human- or primate-derived framework regions at least one peptide with an amino
acid sequence selected from the group of sequences consisting of SEQ ID NO: 2 representing
CDR2 and SEQ ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR)
and SEQ ID NO: 4 representing CDR1 of the Light Chain Variable Region (LCVR).
[0089] Also disclosed is a humanized antibody or a fragment thereof, wherein said humanized
antibody comprises integrated into human- or primate-derived heavy chain framework
regions at least one peptide with an amino acid sequence selected from the group of
sequences consisting of SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing
CDR3 of the Heavy Chain Variable Region (HCVR). Also disclosed is a humanized antibody
or a fragment thereof, wherein said humanized antibody comprises integrated into human-
or primate-derived light chain framework regions a peptide with an amino acid sequence
of SEQ ID NO: 4 representing CDR1 of the Light Chain Variable Region (LCVR).
[0090] Also disclosed is a Light Chain Variable Region (LCVR) comprising integrated into
human- or primate-derived framework regions at least one peptide with an amino acid
sequence of SEQ ID NO: 4 representing CDR1 of the Light Chain Variable Region (LCVR).
[0091] Also disclosed is a Heavy Chain Variable Region (HCVR) comprising integrated into
human- or primate-derived framework regions at least one peptide with an amino acid
sequence selected from the group of sequences consisting of SEQ ID NO: 2 representing
CDR2 and SEQ ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR).
[0092] Also disclosed is a humanized antibody or a fragment thereof, which comprises integrated
into human- or primate-derived framework regions at least two peptides, which peptides
are different and exhibit an amino acid sequence selected from the group of sequences
consisting of SEQ ID NO:1 representing CDR1, SEQ ID NO: 2 representing CDR2 and SEQ
ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR) and SEQ ID NO:
4 representing CDR1, SEQ ID NO: 5 representing CDR2 and SEQ ID NO: 6 representing
CDR3 of the Light Chain Variable Region (LCVR) wherein the same CDR cannot be present
twice in the antibody. In particular, if the at least two CDRs present are both CDRs
of the Light Chain Variable Region (LCVR), at least on of said CDRs must be CDR1 represented
by SEQ ID NO: 4.
[0093] Also disclosed is a humanized antibody or a fragment thereof comprising integrated
into human- or primate-derived heavy chain framework regions at least two peptides
with an amino acid sequence selected from the group of sequences consisting of SEQ
ID NO: 1 representing CDR1, SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing
CDR3 of the Heavy Chain Variable Region (HCVR), but particularly a humanized antibody
or a fragment thereof wherein the same CDR cannot be present twice in the antibody.
[0094] Also disclosed is a Heavy Chain Variable Region (HCVR) comprising integrated into
human- or primate-derived heavy chain framework regions at least two peptides with
an amino acid sequence selected from the group of sequences consisting of SEQ ID NO:
1 representing CDR1, SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing
CDR3 of the Heavy Chain Variable Region (HCVR).
[0095] Also disclosed is a humanized antibody or a fragment thereof, comprising integrated
into human- or primate-derived light chain framework regions at least two peptides
with an amino acid sequence selected from the group of sequences consisting of SEQ
ID NO: 4 representing CDR1, SEQ ID NO: 5 representing CDR2 and SEQ ID NO: 6 representing
CDR3 of the Light Chain Variable Region (LCVR).
[0096] Also disclosed is a Light Chain Variable Region (LCVR), which has integrated into
human- or primate-derived light chain framework regions at least two peptides with
an amino acid sequence selected from the group of sequences consisting of SEQ ID NO:
4 representing CDR1, SEQ ID NO: 5 representing CDR2 and SEQ ID NO: 6 representing
CDR3 of the Light Chain Variable Region (LCVR), wherein the same CDR cannot be present
twice in the antibody and, in particular, at least on of said CDRs must be CDR1 represented
by SEQ ID NO: 4.
[0097] Also disclosed is a humanized antibody or a fragment thereof, comprising integrated
into human- or primate-derived heavy chain framework regions peptides with an amino
acid sequence of SEQ ID NO: 1 representing CDR1, SEQ ID NO: 2 representing CDR2 and
SEQ ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR), particularly
in the order indicated above.
[0098] Also disclosed is a Heavy Chain Variable Region (HCVR) comprising integrated into
human- or primate-derived heavy chain framework regions peptides with an amino acid
sequence of SEQ ID NO: 1 representing CDR1, SEQ ID NO: 2 representing CDR2 and SEQ
ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR), particularly
in the order indicated above.
[0099] Also disclosed is a humanized antibody or a fragment thereof comprising integrated
into human- or primate-derived light chain framework regions peptides with an amino
acid sequence of SEQ ID NO: 4 representing CDR1, SEQ ID NO: 5 representing CDR2 and
SEQ ID NO: 6 representing CDR3 of the Light Chain Variable Region (LCVR), particularly
in the order indicated above.
[0100] Also disclosed is a Light Chain Variable Region (LCVR) comprising integrated into
human- or primate-derived light chain framework regions peptides with an amino acid
sequence of SEQ ID NO: 4 representing CDR1, SEQ ID NO: 5 representing CDR2 and SEQ
ID NO: 6 representing CDR3 of the Light Chain Variable Region (LCVR), particularly
in the order indicated above.
[0101] Also disclosed is a humanized antibody or a fragment thereof, which comprises integrated
into human- or primate-derived framework regions at least three peptides with an amino
acid sequence selected from the group of sequences consisting of SEQ ID NO: 1 representing
CDR1 , SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3 representing CDR3 of the Heavy
Chain Variable Region (HCVR) and SEQ ID NO: 4 representing CDR1, SEQ ID NO: 5 representing
CDR2 and SEQ ID NO: 6 representing CDR3 of the Light Chain Variable Region (LCVR),
but particularly a humanized antibody or a fragment thereof wherein the same CDR cannot
be present twice in the antibody.
[0102] Also disclosed is a humanized antibody or a fragment thereof, which antibody comprises
integrated into human- or primate-derived framework regions at least four peptides
with an amino acid sequence selected from the group of sequences consisting of SEQ
ID NO: 1 representing CDR1, SEQ ID NO: 2 representing CDR2 and SEQ ID NO:3 representing
CDR3 of the Heavy Chain Variable Region (HCVR) and SEQ ID NO: 4 representing CDR1,
SEQ ID NO: 5 representing CDR2 and SEQ ID NO: 6 representing CDR3 of the Light Chain
Variable Region (LCVR), but particularly a humanized antibody or a fragment thereof
wherein the same CDR cannot be present twice in the antibody.
[0103] Also disclosed is a humanized antibody or a fragment thereof, which comprises integrated
into human- or primate-derived framework regions at least five peptides with an amino
acid sequence selected from the group of sequences consisting of SEQ ID NO: 1 representing
CDR1, SEQ ID NO: 2 representing CDR2 and SEQ ID NO:3 representing CDR3 of the Heavy
Chain Variable Region (HCVR) and SEQ ID NO: 4 representing CDR1, SEQ ID NO: 5 representing
CDR2 and SEQ ID NO: 6 representing CDR3 of the Light Chain Variable Region (LCVR),
but particularly a humanized antibody or a fragment thereof wherein the same CDR cannot
be present twice in the antibody.
[0104] Also disclosed is a humanized antibody or a fragment thereof, which comprises integrated
into human- or primate-derived framework regions peptides with an amino acid sequence
of SEQ ID NO: 1 representing CDR1, SEQ ID NO: 2 representing CDR2 and SEQ ID NO: 3
representing CDR3 of the Heavy Chain Variable Region (HCVR) and SEQ ID NO: 4 representing
CDR1, SEQ ID NO: 5 representing CDR2 and SEQ ID NO: 6 representing CDR3 of the Light
Chain Variable Region (LCVR).
[0105] Also disclosed is a antibody, a Heavy Chain Variable Region (HCVR), or a fragment
thereof, wherein said humanized antibody, Heavy Chain Variable Region (HCVR) or fragment
thereof comprises integrated into human- or primate-derived heavy chain framework
regions at least a peptide with an amino acid sequence of SEQ ID NO: 2 representing
CDR2 of the Heavy Chain Variable Region (HCVR).
[0106] Also disclosed is a humanized antibody, a Heavy Chain Variable Region (HCVR) or a
fragment thereof, wherein said humanized antibody, Heavy Chain Variable Region (HCVR)
or fragment thereof comprises integrated into human- or primate-derived heavy chain
framework regions at least a peptide with an amino acid sequence of SEQ ID NO: 3 representing
CDR3 of the Heavy Chain Variable Region (HCVR).
[0107] Also disclosed is humanized antibody, Heavy Chain Variable Region (HCVR) or a fragment
thereof, which antibody, Heavy Chain Variable Region (HCVR) or fragment thereof comprises
integrated into human- or primate-derived heavy chain framework regions at least two
peptides with an amino acid sequence of SEQ ID NO: 1 representing CDR1 and SEQ ID
NO: 2 representing CDR2 of the Heavy Chain Variable Region (HCVR).
[0108] Also disclosed is a humanized antibody, a Heavy Chain Variable Region (HCVR) or a
fragment thereof, which antibody, Heavy Chain Variable Region (HCVR) or fragment thereof
comprises integrated into human- or primate-derived heavy chain framework regions
at least two peptides with an amino acid sequence of SEQ ID NO: 1 representing CDR1
and SEQ ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR).
[0109] Also disclosed is a humanized antibody, a Heavy Chain Variable Region (HCVR) or a
fragment thereof, which antibody, Heavy Chain Variable Region (HCVR) or fragment thereof
comprises integrated into human- or primate-derived heavy chain framework regions
at least two peptides with an amino acid sequence of SEQ ID NO: 2 representing CDR2
and SEQ ID NO: 3 representing CDR3 of the Heavy Chain Variable Region (HCVR).
[0110] Also disclosed is a humanized antibody, a Light Chain Variable Region (LCVR) or a
fragment thereof, which antibody, Light Chain Variable Region (LCVR) or fragment thereof
comprises integrated into human- or primate-derived heavy chain framework regions
at least two peptides with an amino acid sequence of SEQ ID NO: 4 representing CDR1
and SEQ ID NO: 5 representing CDR2 of the Light Chain Variable Region (LCVR).
[0111] Also disclosed is a humanized antibody, a Light Chain Variable Region (LCVR) or a
fragment thereof, which antibody, Light Chain Variable Region (LCVR) or fragment thereof
comprises integrated into human- or primate-derived heavy chain framework regions
at least two peptides with an amino acid sequence of SEQ ID NO: 4 representing CDR1
and SEQ ID NO: 6 representing CDR3 of the Light Chain Variable Region (LCVR).
[0112] Also disclosed is a humanized antibody or a fragment thereof, wherein both the Heavy
Chain Variable Region (HCVR) and the Light Chain Variable Region (LCVR) of the mouse
C2 antibody each contributes at least one of its CDR regions to the at least two CDR
regions of the humanized antibody. The resulting humanized antibody or a fragment
thereof thus may comprise
- at least an amino acid sequence of SEQ ID NO: 1 representing CDR1 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 4 representing CDR1 (LCVR);
- at least an amino acid sequence of SEQ ID NO: 2 representing CDR2 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 4 representing CDR1 (LCVR);
- at least an amino acid sequence of SEQ ID NO: 3 representing CDR3 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 4 representing CDR1 (LCVR);
- at least an amino acid sequence of SEQ ID NO: 1 representing CDR1 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 5 representing CDR2 (LCVR);
- at least an amino acid sequence of SEQ ID NO: 2 representing CDR2 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 5 representing CDR2 (LCVR);
- at least an amino acid sequence of SEQ ID NO:2 representing CDR2 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 6 representing CDR3 (LCVR);
- at least an amino acid sequence of SEQ ID NO:1 representing CDR1 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 6 representing CDR3 (LCVR);
- at least an amino acid sequence of SEQ ID NO: 3 representing CDR3 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 5 representing CDR2 (LCVR);
- at least an amino acid sequence of SEQ ID NO: 3 representing CDR3 (HCVR) in combination
with an amino acid sequence of SEQ ID NO: 6 representing CDR3 (LCVR).
[0113] The humanized antibody of the invention may comprise a light chain and/or a heavy
chain constant region of human or primate origin.
[0114] In a further embodiment, the invention relates to a humanized antibody or a fragment
thereof, wherein at least one, particularly at least one but not more than 5, more
particularly at least one but not more than 4, even more particularly at least one
but not more than 3, but especially at least one but not more than 2, of the amino
acids representative of the light chain and/or heavy chain CDR regions as given in
SEQ ID NOs: 1 - 6 is changed through a conservative substitution such that the antibody
maintains its full functionality.
[0115] In particular, the invention relates to a humanized antibody or a fragment thereof,
wherein in CDR2 of the light chain variable region (LCVR) as given in SEQ ID NO: 5,
the Lys at Kabat position 50 is replaced by an amino acid residue selected from the
group consisting of Arg, Gln and Glu, particularly by Arg.
[0116] In particular, the invention relates to a light chain variable region (LCVR) wherein
in CDR2 as given in SEQ ID NO: 5, the Lys at Kabat position 50 is replaced by an amino
acid residue selected from the group consisting of Arg, Gln and Glu, particularly
by Arg.
In another embodiment, the invention relates to a humanized antibody or a fragment
thereof, wherein in CDR2 of the light chain variable region (LCVR) as given in SEQ
ID NO: 5, the Ser at Kabat position 53 is replaced by an amino acid residue selected
from the group consisting of Asn or Thr, but particularly by Asn.
[0117] In particular, the invention relates to a light chain variable region (LCVR) wherein
in CDR2 as given in SEQ ID NO: 5, the Ser at Kabat position 53 is replaced by an amino
acid residue selected from the group consisting of Asn or Thr, but particularly by
Asn.
[0118] In one embodiment of the invention, a humanized antibody or a fragment thereof is
provided, wherein the Heavy Chain Variable Region (HCVR) has an amino acid sequence
that is 90%, particularly 95%, more particularly 98% identical to the sequence given
in SEQ ID NO: 15 and 16, respectively.
[0119] In another embodiment of the invention, a humanized antibody or a fragment thereof
is provided, wherein the Light Chain Variable Region (LCVR) has an amino acid sequence
that is 90%, particularly 95%, more particularly 98% identical to the sequence given
in SEQ ID NO: 12 and 13, respectively.
In still another embodiment of the invention, a humanized antibody or a fragment thereof
is provided, wherein at least two, but especially three, of the CDR regions of the
Heavy Chain Variable Region (HCVR) have an amino acid sequence that is 90%, particularly
95%, more particularly 98% identical to the corresponding CDR region as given in SEQ
ID NO: 1 - 3.
[0120] In a further embodiment of the invention, a humanized antibody or a fragment thereof
is provided, wherein at least two, but especially three, of the CDR regions of the
Light Chain Variable Region (LCVR) have an amino acid sequence that is 90%, particularly
95%, more particularly 98% identical to the corresponding CDR region as given in SEQ
ID NO: 4 - 6.
[0121] In still another embodiment, the invention relates to a humanized antibody or a fragment
thereof according to the present invention as described herein before wherein the
Heavy Chain Variable Region (HCVR) has an amino acid sequence that is 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence given in SEQ ID NO:
15 and 16, respectively.
[0122] In still another embodiment, the invention relates to a humanized antibody or a fragment
thereof according to the present invention as described herein before wherein the
Light Chain Variable Region (LCVR) has an amino acid sequence that is 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence given in SEQ ID NO:
12 and 13, respectively.
[0123] In still another embodiment, the invention relates to a humanized antibody or a fragment
thereof according to the present invention as described herein before, wherein at
least one, particularly at least two, but especially three, of the CDR regions of
the Heavy Chain Variable Region (HCVR) have an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding CDR region
as given in SEQ ID NO: 1 - 3.
[0124] In still another embodiment, the invention relates to a humanized antibody or a fragment
thereof according to the present invention as described herein before, wherein at
least one, particularly at least two, but especially three, of the CDR regions of
the Light Chain Variable Region (LCVR) have an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding CDR region
as given in SEQ ID NO: 4 - 6.
[0125] In still another embodiment, the invention relates to a humanized antibody according
to the present invention and as described herein before, wherein at least one of the
amino acids representative of the acceptor framework sequences obtained from human
germline V
H and V
K sequences, respectively is changed through a substitution to an amino acid from the
corresponding region of murine antibody ACI-01-Ab7C2 or a substitution conservative
thereto.
[0126] In particular, the invention relates to a Heavy Chain Variable Region and to a humanized
antibody comprising this Heavy Chain Variable Region, respectively, wherein the Trp
in Kabat position 47 in the acceptor framework sequence obtained from human germline
V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe, particularly
Leu and Ile, but especially Leu such as shown in SEQ ID NO: 15.
[0127] The invention further relates to a Heavy Chain Variable Region and to a humanized
antibody comprising this Heavy Chain Variable Region, respectively, wherein the Arg
in Kabat position 94 in the acceptor framework sequence obtained from human germline
V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Ser and Thr, but especially by Ser such as shown in SEQ ID
NO: 15.
[0128] In still another embodiment, the invention relates to a Heavy Chain Variable Region
and to a humanized antibody comprising this Heavy Chain Variable Region, respectively,
wherein the Trp in Kabat position 47 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe, particularly
Leu and Ile, but especially Leu and the Arg in Kabat position 94 is replaced by an
amino acid selected from the group consisting of Ser and Thr, but especially by Ser
such as shown in SEQ ID NO: 15.
[0129] The invention further relates to a Light Chain Variable Region and to a humanized
antibody comprising this Light Chain Variable Region, respectively, wherein the Gln
in Kabat position 45 in the acceptor framework sequence obtained from human germline
V
K sequences of KABAT subgroup V
KII of the Light Chain Variable Region is replaced by an amino acid selected from the
group consisting of Lys, Arg, Gln, and Asn, particularly by Lys and Arg, but especially
by Lys.
[0130] The invention further relates to a Light Chain Variable Region and to a humanized
antibody comprising this Light Chain Variable Region, respectively, wherein the Tyr
in Kabat position 87 in the acceptor framework sequence obtained from human germline
V
K sequences of KABAT subgroup V
KII of the Light Chain Variable Region is replaced by an amino acid selected from the
group consisting of Phe, Leu, Val, Ile, and Ala, particularly by Leu and Phe, but
especially by Phe.
[0131] The invention further relates to a Light Chain Variable Region and to a humanized
antibody comprising this Light Chain Variable Region, respectively, wherein the Lys
in Kabat position 50 in the CDR2 region obtained from a mouse monoclonal antibody,
particularly murine antibody ACI-01-Ab7C2, such as shown in SEQ ID NO: 12 is replaced
by an Arg.
[0132] In still another embodiment, the invention relates to a Light Chain Variable Region
and to a humanized antibody comprising this Light Chain Variable Region, respectively,
wherein the Asn in Kabat position 53 in the CDR2 region obtained from a mouse monoclonal
antibody, particularly murine antibody ACI-01-Ab7C2, such as shown in SEQ ID NO: 12
is replaced by Ser.
[0133] In still another embodiment, the invention relates to a humanized antibody, wherein
the Trp in Kabat position 47 in the acceptor framework sequence obtained from human
germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe, particularly
Leu and Ile, but especially Leu and the Arg in Kabat position 94 in the acceptor framework
sequence obtained from human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Ser and Thr, but especially by Ser as shown in SEQ ID NO:
15, and the Tyr in Kabat position 87 in the acceptor framework sequence obtained from
human germline V
K sequences of KABAT subgroup V
KII of the Light Chain Variable Region is replaced by an amino acid selected from the
group consisting of Phe, Leu, Val, Ile, and Ala, particularly by Leu and Phe, but
especially by Phe.
[0134] In still another embodiment, the invention relates to a Heavy Chain Variable Region
and to a humanized antibody comprising this Heavy Chain Variable Region, respectively,
wherein the Trp in Kabat position 47 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region as shown in SEQ ID NO: 15 is replaced by Leu.
[0135] In still another embodiment, the invention relates to a Heavy Chain Variable Region
and to a humanized antibody comprising this Heavy Chain Variable Region, respectively,
wherein the Arg in Kabat position 94 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by Ser such as shown in SEQ ID
NO: 15.
[0136] In still another embodiment, the invention relates to a Heavy Chain Variable Region
and to a humanized antibody comprising this Heavy Chain Variable Region, respectively,,
wherein the Trp in Kabat position 47 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by Leu and Ile, but especially
Leu and the Arg in Kabat position 94 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by Ser such as shown in SEQ ID
NO: 15.
[0137] In still another embodiment, the invention relates to a Light Chain Variable Region
and to a humanized antibody comprising this Heavy Chain Variable Region, respectively,
wherein the Tyr in Kabat position 87 in the acceptor framework sequence obtained from
human germline V
K sequences of KABAT subgroup V
KII of the Light Chain Variable Region is replaced by Phe.
[0138] In still another embodiment, the invention relates to a Heavy Chain Variable Region
and to a humanized antibody comprising this Heavy Chain Variable Region, respectively,,
wherein the Trp in Kabat position 47 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by Leu and Ile, but especially
Leu and the Arg in Kabat position 94 in the acceptor framework sequence obtained from
human germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by Ser such as shown in SEQ ID
NO: 15 and the Tyr in Kabat position 87 in the acceptor framework sequence obtained
from human germline V
K sequences of KABAT subgroup V
KII of the Light Chain Variable Region is replaced by Phe..
[0139] In one embodiment, the invention relates to a Heavy Chain Variable Region and to
a humanized antibody comprising this Heavy Chain Variable Region, respectively, wherein
the Trp in Kabat position 47 in the acceptor framework sequence obtained from human
germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe, particularly
Leu and Ile, but especially Leu and the Arg in Kabat position 94 is replaced by an
amino acid selected from the group consisting of Ser and Thr, but especially by Ser
such as shown in SEQ ID NO: 15 and wherein the Lys in Kabat position 50 in the CDR2
region obtained from a mouse monoclonal antibody, particularly murine antibody ACI-01-Ab7C2,
is replaced by an amino acid selected from the group consisting of Arg, Gln, His,
and Asn, but especially by Arg.
[0140] In one embodiment, the invention relates to a Heavy Chain Variable Region and to
a humanized antibody comprising this Heavy Chain Variable Region, respectively, wherein
the Trp in Kabat position 47 in the acceptor framework sequence obtained from human
germline V
H sequences of KABAT subgroup V
HIII of the Heavy Chain Variable Region is replaced by an amino acid selected from
the group consisting of Leu, norleucine, Ile, Val, Met, Ala, and Phe, particularly
Leu and Ile, but especially Leu and the Arg in Kabat position 94 is replaced by an
amino acid selected from the group consisting of Ser and Thr, but especially by Ser
such as shown in SEQ ID NO: 15 and wherein the Asn in Kabat position 53 in the CDR2
region obtained from a mouse monoclonal antibody, particularly murine antibody ACI-01-Ab7C2,
is replaced by an amino acid selected from the group consisting of Ala, Val, Leu,
Ser and Ile; but especially Ser.The light chain variable region of SEQ ID NO: 12 is
disclosed. In another specific embodiment of the invention, a humanized antibody is
provided, which comprises the light chain variable region of SEQ ID NO: 12.
[0141] Disclosed is the light chain variable region including signal sequences as shown
in SEQ ID NO: 13.
[0142] In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the complete light chain variable region including signal sequences
as shown in SEQ ID NO: 13.
[0143] In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the light chain variable region of SEQ ID NO: 12 and the light chain
constant region of SEQ ID NO: 14.
[0144] In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the complete light chain variable region of SEQ ID NO: 13 and the
light chain constant region of SEQ ID NO: 14. Disclosed is also the heavy chain variable
region of SEQ ID NO: 15.
[0145] In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the heavy chain variable region of SEQ ID NO: 15. Disclosed is the
heavy chain variable region including signal sequences as shown in SEQ ID NO: 16.
[0146] In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the complete heavy chain variable region including signal sequences
as shown in SEQ ID NO: 16.
In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the heavy chain variable region of SEQ ID NO: 15 and the heavy chain
constant region of SEQ ID NO: 17.
In another specific embodiment of the invention, a humanized antibody is provided,
which comprises the heavy chain variable region of SEQ ID NO: 16 and the heavy chain
constant region of SEQ ID NO: 17.
In one embodiment the humanized antibody according to the invention and as described
herein, upon co-incubation with an A
β monomeric peptide having at least 30, particularly at least 35, more particularly
at least 38, even more particularly at least 40 amino acid residues and/or an A
β polymeric soluble amyloid peptide comprising a plurality of said A
β monomeric units, but especially with an A
β1-42 monomeric and/or an A
β polymeric soluble amyloid peptide comprising a plurality of said A
β1-42 monomeric units, particularly at a molar concentration ratio of antibody to A
β1-42 of up to 1:1000, particularly of up to 1:500, more particularly of up to 1:300,
even more particularly of up to 1:200, but especially at a molar concentration ratio
of between 1:10 and 1:100, inhibits the aggregation of the A
β monomers to high molecular polymeric fibrils.
[0147] In particular, the co-incubation of the antibody according to the invention with
amyloid monomeric and/or polymeric soluble amyloid peptides is carried out for 24
hours to 60 hours, particularly for 30 hours to 50 hours, more particularly for 48
hours, but especially 24 hours, at a temperature of between 28°C and 40°C, particularly
of between 32°C and 38°C, more particularly at 37°C.
[0148] In a specific aspect co-incubation with amyloid monomeric and/or polymeric soluble
amyloid peptides is accomplished for 24 hours at a temperature of 37°C.
[0149] In particular, the humanized antibody according to the invention including any functional
parts thereof binds to A
β1-42 monomeric peptide and/or A
β polymeric soluble amyloid peptide comprising a plurality of said A
β1-42 monomeric units and, upon co-incubation with A
β1-42 monomeric peptide and/or A
β polymeric soluble amyloid peptide comprising a plurality of said A
β1-42 monomeric units inhibits the aggregation of the A
β monomers and/or polymers to high molecular polymeric fibrils.
[0150] In one embodiment, the humanized antibody according to the invention including any
functional parts thereof inhibits the aggregation of the A
β monomers and/or A
β soluble polymers comprising a plurality of said A
β monomeric units to high molecular polymeric fibrils by at least 50%, particularly
by at least 60%, particularly by at least 65%, more particularly by at least 75%,
even more particularly by at least 80%, but especially by at least 85%-90%, or more
as compared to the respective amyloid peptide monomers incubated in buffer (control),
at a molar concentration ratio of antibody to A
β1-42 of up to 1:1000, particularly at a molar concentration ratio of between 1:10
and 1:100, but especially at a molar concentration ratio of 1:10.
[0151] In a specific embodiment of the invention, the humanized antibody according to the
invention including any functional parts thereof inhibits the aggregation of the A
β monomers and/or A
β soluble polymers comprising a plurality of said A
β monomeric units to high molecular polymeric fibrils by at least 30% at a molar concentration
ratio of antibody to A
β1-42 of 1:100.
[0152] In another specific embodiment of the invention, the humanized antibody according
to the invention including any functional parts thereof inhibits the aggregation of
the A
β monomers and/or A
β soluble polymers comprising a plurality of said A
β monomeric units to high molecular polymeric fibrils by at least 80% at a molar concentration
ratio of antibody to A
β1-42 of 1:10.
[0153] Binding of the antibodies according to the invention and as described herein to amyloidogenic
monomeric and/or polymeric peptides but, particularly, to the amyloid form (1-42)
leads to inhibition of the aggregation of monomeric and/or polymeric amyloidogenic
peptides to high molecular fibrils or filaments. Through the inhibition of the aggregation
of amyloidogenic monomeric and/or polymeric peptides the antibodies according to the
present invention are capable of preventing or slowing down the formation of amyloid
plaques, particularly the amyloid form (1-42), which is know to become insoluble by
change of secondary conformation and to be the major part of amyloid plaques in brains
of diseased animals or humans.
[0154] The aggregation inhibition potential of the antibody according to the invention may
be determined by any suitable method known in the art, particularly by density-gradient
ultracentrifugation followed by a SDS-PAGE sedimentation analysis on a preformed gradient
and/or by a thioflavin T (Th-T) fluorescent assay.
[0155] In one embodiment, the invention relates to a humanized antibody as described herein
including any functional parts thereof, which antibody, upon co-incubation, particularly
at a molar concentration ratio of between 1:5 and 1:1000, particularly of between
1:10 and 1:500, more particularly at a ratio of 1:10 to 1:300, even more particularly
at a ratio of between 1:10 and 1:100, with preformed high molecular polymeric amyloid
fibrils or filaments formed by the aggregation of A
β monomeric peptides having at least 30, particularly at least 35, more particularly
at least 38, even more particularly at least 40 amino acid residues and, but especially
A
β1-42 monomeric peptides, is capable of disaggregating the preformed polymeric fibrils
or filaments by at least 20%, particularly by at least 30%, more particularly by at
least 35%%, even more particularly by at least 40%, but especially by at least 50%
or more.
[0156] In a specific embodiment of the invention, the aggregation inhibition and the disaggregation
potential of the antibody, respectively, is determined by density-gradient ultracentrifugation
followed by a SDS-PAGE sedimentation analysis on a preformed gradient.
[0157] In another specific embodiment of the invention, the aggregation inhibition and the
disaggregation potential of the antibody, respectively, is determined by thioflavin
T (Th-T) fluorescent assay.
[0158] In another specific embodiment, the antibody according to the invention is co-incubated
with amyloid preformed high molecular polymeric amyloid fibrils or filaments for 12
hours to 36 hours, particularly for 18 hours to 30 hours, more particularly for 24
hours at a temperature of between 28°C and 40°C, particularly of between 32°C and
38°C, more particularly at 37°C.
[0159] In particular, the co-incubation with preformed high molecular polymeric amyloid
fibrils or filaments is done for 24 hours at a temperature of 37°C.
[0160] In a specific embodiment of the invention, the humanized antibody according to the
invention including any functional parts thereof is capable of disaggregating the
preformed polymeric fibrils or filaments by at least 24% at a molar concentration
ratio of antibody to A
β1-42 of 1:100.
[0161] In another specific embodiment of the invention, the humanized antibody according
to the invention including any functional parts thereof is capable of disaggregating
the preformed polymeric fibrils or filaments by at least 32% at a molar concentration
ratio of antibody to A
β1-42 of 1:10.
[0162] Through the disaggregation of amyloidogenic polymeric fibrils or filaments the antibodies
according to the present invention are capable of preventing or slowing down the formation
of amyloid plaques which leads to an alleviation of the symptoms associated with the
disease and a delay or reversal of its progression.
[0163] Accordingly, it is a further embodiment of the invention to provide a humanized antibody,
including any functional parts thereof as described herein, which antibody is capable
of decreasing the total amount of A
β in the brain of an animal, particularly a mammal, but especially a human suffering
from a disease or condition leading to increased concentration of A
β in the brain.
[0164] In another embodiment, the invention relates to a humanized antibody according to
the invention and as described herein before, which antibody is bi-effective in that
it exhibits both an aggregation inhibition property as well as a disaggregation property,
particularly paired with a high degree of conformational sensitivity.
[0165] In particular, the invention relates to a humanized antibody or a fragment thereof
according to the invention and as described herein before, which antibody, upon co-incubation
with amyloid monomeric and/or polymeric soluble amyloid peptides, particularly with
β-amyloid monomeric peptides such as, for example, A
β monomeric peptides 1-39; 1-40, 1-41, or 1-42, and/or a polymeric soluble
β-amyloid peptide comprising a plurality of said A
β monomeric units, but especially with an A
β1-42 monomeric and/or an A
β polymeric soluble amyloid peptide comprising a plurality of said A
β1-42 monomeric units,, inhibits the aggregation of the A
β monomers into high molecular polymeric fibrils or filaments and, in addition, upon
co-incubation with preformed high molecular polymeric amyloid fibrils or filaments
formed by the aggregation of amyloid monomeric peptides, particularly
β-amyloid monomeric peptides such as, for example, A
β monomeric peptides 1-39; 1-40, 1-41, or 1-42, but especially A
β1-42 monomeric peptides, is capable of disaggregating the preformed polymeric fibrils
or filaments.
[0166] In another aspect, the invention relates to a humanized antibody or a fragment thereof
according to the present invention and as described herein before, which antibody
is capable of inducing a transition of the
β-sheet conformation towards an
α-helix and/or a random coil conformation, but particularly a random coil conformation,
even more particularly a random coil conformation at a given location in the molecule,
especially in the environment of Tyr 10 and Val12 of the A
β protein, which leads to an increase of the random coil conformation at the expense
of the
β-sheet conformation and an improved solubilization of the preformed high molecular
polymeric amyloid fibrils or filaments. In particular the decrease of the
β-sheet conformation amounts to at least 30%, particularly to at least 35%, and more
particularly to at least 40% and more as compared to the respective preformed amyloid
polymeric fibrils or filaments incubated in buffer (control).
[0167] The antibody's potential in inducing a transition in the secondary structure is determined
by solid state 13C NMR spectroscopy but, in particular, by measuring the integral
intensities of the conformations of Tyr 10 and Val 12 C
β in the A
β1-42 peptide. A humanized antibody or a fragment as described herein before, is provided
comprising at least one light chain or a fragment thereof or at least one heavy chain
or a fragment thereof, wherein said antibody or fragment binds to an A
β monomer with a high binding affinity with a K
D in a range of between at least about 1 x 10
-7 M to at least about 1 x 10
-12 M, particularly of at least about 1 x 10
-8 M to at least about 1 x 10
-11 M, more particularly of at least about 1 x 10
-9 M to at least about 1 x 10
-10 M, even more particularly of at least about 1 x 10
-8 M to at least about 2 x 10
-8.M
, but, preferably, does not show any significant cross-reactivity with amyloid precursor
protein (APP). A humanized antibody or a fragment as described herein before, is provided
comprising at least one light chain or a fragment thereof or at least one heavy chain
or a fragment thereof, wherein said antibody or fragment binds to an A
β fiber, fibril or filament with a high binding affinity with a K
D in a range of between at least about 1 x 10
-7 M to at least about 1 x 10
-12 M, particularly of at least about 1 x 10
-8 M to at least about 1 x 10
-11 M, more particularly of at least about 1 x 10
-9 M to at least about 1 x 10
-10 M, even more particularly of at least about 2 x 10
-9 M to at least about 5 x 10
-9 M, but, preferably, does not show any significant cross-reactivity with amyloid precursor
protein (APP).
[0168] In another embodiment, the humanized antibody according to the invention and as described
herein before or a fragment thereof, exhibits an binding affinity to an A
β fiber, fibril or filament which is at least 2 times, particularly at least 4 times,
particularly at least 10 times, particularly at least 15 times, more particularly
at least 20 times, but especially at least 25 times higher than the binding affinity
to an A
β monomer.
[0169] In still another embodiment, a humanized antibody or a fragment thereof according
to the present invention and as described herein before, substantially binds to aggregated
A
β, including A
β plaques, in the mammalian, particularly the human brain but, preferably, does not
show any significant cross-reactivity with amyloid precursor protein (APP). In another
aspect of the invention, a humanized antibody or a fragment thereof according to the
present invention and as described herein before, substantially binds to soluble polymeric
amyloid, particularly amyloid
β (A
β), including A
β monomers, in the mammalian, particularly the human brain but, preferably, does not
show any significant cross-reactivity with amyloid precursor protein (APP).
[0170] Further provided is a humanized antibody or a fragment thereof according to the invention
and as described herein before, which antibody significantly reduces A
β plaque burden in the mammalian, particularly the human brain. This can be achieved
by either binding of the antibody to the plaque or by shifting the equilibrium between
amyloid, particularly amyloid
β (A
β), in its insoluble and aggregated state towards its soluble form by disaggregating
fibers to soluble poly- and monomeric forms by inducing a shift in conformation and
binding and stabilizing the disaggregated and solubilized amyloid forms, particularly
amyloid
β (A
β) forms, in the tissue and/or body fluids, particularly the brain. Through the activity
of the antibody according to the invention the peripheral clearing and catabolism
is thus favored rather than deposition within the tissue and/or body fluids, particularly
the brain. The beneficial effect of the antibody according to the invention can thus
be obtained without binding of the antibody to the plaque.
[0171] Through this stabilizing activity, the antibody according to the invention is able
to neutralize the toxic effects of the polymeric and less aggregated soluble amyloid
protein, particularly amyloid
β (A
β) protein, in the tissue and/or body fluids. In a specific embodiment of the invention
the antibody according to the invention may thus achieve its beneficial effects without
necessarily binding aggregated amyloid beta in the brain. A humanized antibody or
a fragment thereof as described herein before, is provided comprising at least one
light chain or a fragment thereof or at least one heavy chain or a fragment thereof
incorporating at least one, particularly two and more particularly three CDR regions
obtained form a mouse donor antibody, particularly from mouse antibody ACI-01-Ab7C2
(named "mC2" and hC2 for the humanized C2 antibody, throughout the application) deposited
01 December 2005 with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH (DSMZ) in Braunschweig, Mascheroder Weg 1 B, 38124 Braunschweig, under accession
no DSM ACC2750, wherein said antibody or fragment thereof has an affinity to the A
β antigen which is at least 5 times, particularly at least 8 times, more particularly
at least 10 times, but especially at least 15 times higher than that of the mouse
donor antibody.
[0172] The antibody of this invention can be, in one embodiment, a whole antibody (e.g.,
with two full length light chains and two full length heavy chains) of any isotype
and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1 and IgA2); but especially
an antibody of the IgG4 isotype; alternatively, in another embodiment, it can be an
antigen-binding fragment (e.g., Fab, F(ab')
2, and Fv) of a whole antibody.
[0173] The invention thus also relates to antigen-binding fragments of the antibodies described
herein. In one embodiment of the invention, the fragment is selected from the group
consisting of a Fab fragment, a Fab' fragment, a F(ab)
2fragment, and a F
v fragment, including the products of an Fab immunoglobulin expression library and
epitope-binding fragments of any of the antibodies and fragments mentioned above.
[0174] In another embodiment, the antibody or antigen-binding fragment of the invention
is conjugated to polyethylene glycol. In yet another embodiment, the constant region
of the antibody of the invention is modified to reduce at least one constant region-mediated
biological effector function relative to an unmodified antibody. In still another
embodiment, the antibody or antigen-binding fragment of the invention comprises a
Fc region having an altered effector function.
[0175] The invention further relates to a nucleotide molecule comprising a nucleotide sequence
encoding a humanized antibody or a fragment thereof according to the invention and
as disclosed herein before.
[0176] In particular, the invention relates to a nucleotide molecule comprising a nucleotide
sequence encoding a stretch of contiguous amino acid molecules as given in SEQ ID
NO: 2 and 3, respectively, or the complementary sequence, representing the Complementarity
Determining Regions (CDRs) 2 and 3 of the Heavy Chain Variable Region (HCVR).
[0177] More particularly, the invention relates to a nucleotide molecule comprising a nucleotide
sequence encoding a stretch of contiguous amino acid molecules as given in SEQ ID
NO: 4, or the complementary sequence, representing the Complementarity Determining
Regions (CDRs) 1 of the Light Chain Variable Region (LCVR).
[0178] In another aspect a nucleotide molecule is provided comprising a nucleotide sequence
as given in SEQ ID NO: 18 and SEQ ID NO: 19, or the complementary sequence, encoding
the amino acid sequence of CDR 2 and CDR 3, respectively, of the Heavy Chain Variable
Region (HCVR).
[0179] In another aspect a nucleotide molecule is provided comprising a nucleotide sequence
as given in SEQ ID NO: 20, or the complementary sequence, encoding the nucleotide
sequence of CDR 1 of the Light Chain Variable Region (LCVR).
[0180] In another embodiment of the invention the nucleotide molecule of the invention comprises
a nucleotide sequence of SEQ ID NO: 21, or the complementary sequence, encoding the
light chain variable region.
[0181] In another embodiment of the invention the nucleotide molecule of the invention comprises
a nucleotide sequence of SEQ ID NO: 22, or the complementary sequence, encoding the
complete light chain variable region including signal sequences.
[0182] In another embodiment of the invention the nucleotide molecule of the invention comprises
a nucleotide sequence encoding the light chain variable region of SEQ ID NO: 22 and
the light chain constant region of SEQ ID NO: 23.
[0183] In another embodiment of the invention the nucleotide molecule of the invention comprises
a nucleotide sequence of SEQ ID NO: 24 encoding the heavy chain variable region.
[0184] In another embodiment of the invention the nucleotide molecule of the invention comprises
a nucleotide sequence of SEQ ID NO: 25 encoding the complete heavy chain variable
region including signal sequences.
[0185] In another embodiment of the invention a nucleotide molecule is provided comprising
a nucleotide sequence encoding the heavy chain variable region of SEQ ID NO: 25 and
the heavy chain constant region of SEQ ID NO: 26 . The invention also comprises the
complementary strand of said nucleotide molecule.
[0186] Also comprised herein is a nucleotide sequence which hybridizes to one of the above-described
antibody-encoding nucleotide sequences of the invention, particularly to the complementary
strand thereof, either in isolation or as part of larger nucleotide molecule.
[0187] In particular, the invention relates to a nucleotide sequence that hybridizes under
conventional hybridization conditions, particularly under stringent hybridization
conditions, to any of the nucleotide sequences given in SEQ ID NOs: 18-26 and 29 -
32, particularly to the complementary strand thereof.
[0188] In another embodiment of the invention an expression vector is provided comprising
the nucleic acid molecule according to the invention. In another embodiment of the
invention a cell is provided comprising an expression vector comprising the nucleic
acid according to the invention. In still another embodiment, the invention relates
to a composition comprising the antibody according to the invention, but particularly
a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof
according to the invention and as described herein before including any functionally
equivalent antibody or any derivative or functional parts thereof, in a therapeutically
effective amount, in particular a composition which is a pharmaceutical composition
optionally further comprising a pharmaceutically acceptable carrier. In another embodiment
of the invention, said composition comprises the antibody in a therapeutically effective
amount.
[0189] Further comprised by the invention is a mixture comprising a humanized antibody or
a fragment thereof according to the invention including any functional parts thereof,
in a therapeutically effective amount and, optionally, a further biologically active
substance and/or a pharmaceutically acceptable carrier and/or a diluent and/or an
excipient.
[0190] In particular, the invention relates to a mixture, wherein the further biologically
active substance is a compound used in the medication of amyloidosis, a group of diseases
and disorders associated with amyloid or amyloid-like protein such as the A
β protein involved in Alzheimer's disease.
[0191] In another embodiment of the invention, the other biologically active substance or
compound may also be a therapeutic agent that may be used in the treatment of amyloidosis
caused by amyloid
β or may be used in the medication of other neurological disorders.
[0192] The other biologically active substance or compound may exert its biological effect
by the same or a similar mechanism as the antibody according to the invention or by
an unrelated mechanism of action or by a multiplicity of related and/or unrelated
mechanisms of action.
[0193] Generally, the other biologically active compound may include neutron-transmission
enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium-channel
blockers, biogenic amines, benzodiazepine tranquillizers, acetylcholine synthesis,
storage or release enhancers, acetylcholine postsynaptic receptor agonists, monoamine
oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal
anti-inflammatory drugs, antioxidants, and serotonergic receptor antagonists.
[0194] More particularly, the invention relates to a mixture comprising at least one compound
selected from the group consisting of compounds effective against oxidative stress,
anti-apoptotic compounds, metal chelators, inhibitors of DNA repair such as pirenzepin
and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),
α-secretase activators,
β- and γ-secretase inhibitors, tau proteins, neurotransmitter,
β-sheet breakers, attractants for amyloid beta clearing / depleting cellular components,
inhibitors of N-terminal truncated amyloid beta including pyroglutamated amyloid beta
3-42, anti-inflammatory molecules, or cholinesterase inhibitors (ChEIs) such as tacrine,
rivastigmine, donepezil, and/or galantamine, M1 agonists and other drugs including
any amyloid or tau modifying drug and nutritive supplements, and nutritive supplements,
together with an antibody according to the present invention and, optionally, a pharmaceutically
acceptable carrier and/or a diluent and/or an excipient.
[0195] The invention further relates to a mixture, wherein the compound is a cholinesterase
inhibitor (ChEIs), particularly a mixture, wherein the compound is one selected from
the group consisting of tacrine, rivastigmine, donepezil, galantamine, niacin and
memantine.
[0196] In a further embodiment, the mixtures according to the invention may comprise niacin
or memantine together with an antibody according to the present invention and, optionally,
a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.
[0197] In still another embodiment of the invention mixtures are provided that comprise
"atypical antipsychotics" such as, for example clozapine, ziprasidone, risperidone,
aripiprazole or olanzapine for the treatment of positive and negative psychotic symptoms
including hallucinations, delusions, thought disorders (manifested by marked incoherence,
derailment, tangentiality), and bizarre or disorganized behavior, as well as anhedonia,
flattened affect, apathy, and social withdrawal, together with an antibody, particularly
a monoclonal antibody according to the invention, but particularly a chimeric antibody
or a fragment thereof, or a humanized antibody or a fragment thereof according to
the invention and as described herein and, optionally, a pharmaceutically acceptable
carrier and/or a diluent and/or an excipient.
[0198] In a specific embodiment of the invention, the compositions and mixtures according
to the invention comprise the antibody and the biologically active substance, respectively,
in a therapeutically effective amount.
[0199] Other compounds that can be suitably used in mixtures in combination with the antibody
according to the present invention are described in
WO 2004/058258 (see especially pages 16 and 17) including therapeutic drug targets (page 36-39),
alkanesulfonic acids and alkanolsulfuric acids (pages 39-51), cholinesterase inhibitors
(pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal
anti-inflammatory drugs (pages 60-61), antioxidants (pages 61-62), peroxisome proliferators-activated
receptor (PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages 68-75);
amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal
chelators (pages 78-79), anti-psychotics and antidepressants (pages 80-82), nutritional
supplements (pages 83-89) and compounds increasing the availability of biologically
active substances in the brain (see pages 89-93) and prodrugs (pages 93 and 94).
[0200] In another embodiment, the invention relates to a mixture comprising the antibody,
particularly a monoclonal antibody according to the invention, but particularly a
chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof
according to the invention and as described herein before and/or the biologically
active substance in a therapeutically effective amount.
[0201] The invention further relates to the use of a humanized antibody or a fragment thereof
according to the invention and as described herein before and/or a functional part
thereof and/or a pharmaceutical composition, or a mixture comprising said antibody,
for the preparation of a medicament for treating or alleviating the effects of amyloidosis,
a group of diseases and disorders associated with amyloid plaque formation including
secondary amyloidosis and age-related amyloidosis such as diseases including, but
not limited to, neurological disorders such as Alzheimer's Disease (AD), Lewy body
dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch
type); the Guam Parkinson-Dementia complex; as well as other diseases which are based
on or associated with amyloid-like proteins such as progressive supranuclear palsy,
multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia,
ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac amyloidosis;
endocrine tumors, and others, including macular degeneration.
[0202] Also comprised by the present invention is a method for the preparation of a humanized
antibody or a fragment thereof according to the invention and/or a functional part
thereof and/or a pharmaceutical composition, or a mixture comprising said antibody
and/or a functional part thereof, particularly in a therapeutically effective amount,
for use in a method of preventing, treating or alleviating the effects of amyloidosis,
a group of diseases and disorders associated with amyloid plaque formation including
secondary amyloidosis and age-related amyloidosis such as diseases including, but
not limited to, neurological disorders such as Alzheimer's Disease (AD), Lewy body
dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch
type); the Guam Parkinson-Dementia complex; as well as other diseases which are based
on or associated with amyloid-like proteins such as progressive supranuclear palsy,
multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia,
ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac amyloidosis;
endocrine tumors, and others, including macular degeneration comprising formulating
a humanized antibody or a fragment thereof according to the invention in a pharmaceutically
acceptable form.
[0203] Further comprised by the present invention is a humanized antibody and/or a functional
part thereof. or a composition or mixture comprising such an antibody and/or a functional
part thereof for use in a method for preventing, treating or alleviating the effects
of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation
including secondary amyloidosis and age-related amyloidosis such as diseases including,
but not limited to, neurological disorders such as Alzheimer's Disease (AD), Lewy
body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch
type); the Guam Parkinson-Dementia complex; as well as other diseases which are based
on or associated with amyloid-like proteins such as progressive supranuclear palsy,
multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia,
ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac amyloidosis;
endocrine tumors, and others, including macular degeneration comprising administering
the antibody in a therapeutically effective amount to an animal or a human affected
by such a disorder.
[0204] It is also an object of the invention to treat amyloidosis, a group of diseases and
disorders associated with amyloid plaque formation including secondary amyloidosis
and age-related amyloidosis including, but not limited to, neurological disorders
such as Alzheimer's Disease (AD), particularly a disease or condition characterized
by a loss of cognitive memory capacity by administering to an animal, particularly
a mammal or a human, a humanized antibody, particularly a pharmaceutical composition
according to the invention and as described herein.
[0205] In a specific embodiment the invention relates to retaining or increasing cognitive
memory capacity but, particularly, for restoring the cognitive memory capacity of
an animal, particularly a mammal or a human, suffering from memory impairment by administering
to an animal, particularly a mammal or a human, a humanized antibody, particularly
a pharmaceutical composition according to the invention. It is a further object of
the invention to provide a therapeutic composition and treatment of amyloidosis, a
group of diseases and disorders associated with amyloid plaque formation including
secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological
disorders such as Alzheimer's Disease (AD), particularly a disease or condition characterized
by a loss of cognitive memory capacity, using an antibody according to the invention
and as described herein before.
[0206] In particular, the invention relates to the use of a humanized antibody of the invention
in a method for the treatment of an animal, particularly a mammal or a human, suffering
from an amyloid-associated condition characterized by a loss of cognitive memory capacity
leads to the retention of cognitive memory capacity.
[0207] The invention further relates to a method of diagnosis of an amyloid-associated disease
or condition in a patient comprising detecting the immunospecific binding of an antibody
or an active fragment thereof to an epitope of the amyloid protein in a sample or
in situ which includes the steps of
- (a) bringing the sample or a specific body part or body area suspected to contain
the amyloid protein into contact with a humanized antibody or a fragment thereof according
to the invention and/or a functional part thereof, which antibody binds an epitope
of the amyloid protein;
- (b) allowing the antibody and/or a functional part thereof, to bind to the amyloid
protein to form an immunological complex;
- (c) detecting the formation of the immunological complex; and
- (d) correlating the presence or absence of the immunological complex with the presence
or absence of amyloid protein in the sample or specific body part or area.
[0208] The invention also relates to a method of determining the extent of amyloidogenic
plaque burden in a tissue and/or body fluids comprising
- (a) testing a tissue and/or body fluid sample for the presence of amyloid protein
with a humanized antibody or a fragment thereof according to the invention and/or
a functional part thereof;
- (b) determining the amount of antibody bound to the protein; and
- (c) calculating the plaque burden in the tissue or body fluid sample
[0209] In particular, the formation of the immunological complex in step b is determined
such that presence or absence of the immunological complex correlates with presence
or absence of amyloid protein.
[0210] In another embodiment of the invention, a test kit for detection and diagnosis of
amyloid-associated diseases and conditions is provided comprising a humanized antibody
or a fragment thereof according to the invention and/or a functional part thereof.
[0211] In particular, the invention relates to a test kit for detection and diagnosis of
amyloid-associated diseases and conditions comprising a container holding one or more
antibodies according to the present invention, and/or a functional part thereof, and
instructions for using the antibodies for the purpose of binding to amyloid protein
to form an immunological complex and detecting the formation of the immunological
complex such that presence or absence of the immunological complex correlates with
presence or absence of amyloid protein.
[0212] In another aspect, disclosed herein is an antibody comprising a variable region as
recited in SEQ ID NO: 27, or a variant thereof. In one aspect, a cell line expressing
the antibody.
[0213] In another aspect, disclosed herein is an antibody gene comprising a variable region
as recited in SEQ ID NO: 29, or a variant thereof. In one aspect, a cell line expresses
the antibody.
[0214] In another aspect, the invention relates to a method for disaggregating preformed
beta-amyloid fibers, comprising interacting an hC2 antibody with preformed beta-amyloid
fibers.
[0215] In another aspect, the humanized antibody or a fragment thereof of the invention
protects neurons from Abeta-induced degradation.
[0216] In another aspect, the invention relates to preventing Abeta-induced neuron degradation
comprising treating neurons with an effective amount of the humanized antibody or
a fragment thereof according to the invention.
[0217] In another aspect, the invention relates to the use of a humanized antibody or a
fragment thereof according to the invention herein for the preparation of a medicament
for preventing degeneration of neurons upon exposure to Abeta oligomer.
BRIEF DESCRIPTION OF FIGURES AND SEQUENCES
[0218]
Figure 1 (Example 2): Expression Cassette of the mouse light chain variable region
of the Chimeric Antibody
Figure 2 (Example 2): Expression Cassette of the mouse heavy chain variable region
of the Chimeric Antibody
Figure 3 (Example 5.2): Comparison of the mouse heavy chain variable region to the
closest murine germ line sequence
Figure 4 (Example 8): Activity of purified humanized C2 antibodies
Figure 5 (Example 9): Binding activity of antibodies produced by transient expression
of C2 modified CDRL2 constructs in conjunction with C2 chimeric heavy chain, compared
to chimeric antibody C2ChVHAF/ChVK, produced by transient transfection and purified
antibody.
Figure 6 (Example 11): Results of Immunohistochemical Binding Assay with chimeric
antibody AF and humanized antibody H4K1.
Figure 7 (Example 12): Functionality of mC2 on Amyloid fibers
Figure 8 (Example 12): Binding Affinity of humanized C2 in ELISA.
Figure 9 (Example 14): Conformation specific binding of mC2 to different classes of
amyloid protein. Pellet preparation in the legend to this figure refers to Aβ1-42 fibers, supernatant preparation refers to amyloid monomers.
Figure 10: Humanized C2 VK sequences compared to murine sequence and human acceptor
sequences DPK15 AND JK1
Figure 11: Humanized C2 VH sequences compared to murine sequence and human acceptor
sequences DP54 AND JH6
Figure 12: Complete DNA and protein sequence of light chain variable region of C2
humanized antibody, C2HuVK1
Figure 13: Complete DNA and protein sequence of light chain constant region (human
C Kappa) of humanized C2 antibody
Figure 14: Complete DNA and protein sequence of heavy chain constant region (human
IgG4 ser228-pro) of humanized C2 antibody
Figure 15A-C (Example 15): Results of Epitope Mapping experiments
Figure 16 (Example 13): Results of aggregation assay experiments
Figure 17 (Example 13): Results of disaggregation assay experiments
Figure 18: (Example 16): Results of neuroprotection experiments with humanized antibody
C2.
SEQ ID NO: 1 Amino acid sequence of C2 HuVH AF 4 humanized heavy chain variable region
(CDR1)
SEQ ID NO: 2 Amino acid sequence of C2 HuVH AF 4 humanized heavy chain variable region
(CDR2)
SEQ ID NO: 3 Amino acid sequence of C2 HuVH AF 4 humanized heavy chain variable region
(CDR3)
SEQ ID NO: 4 Amino acid sequence of C2 HuVK 1 humanized light chain variable region
(CDR1)
SEQ ID NO: 5 Amino acid sequence of C2 HuVK 1 humanized light chain variable region
(CDR2)
SEQ ID NO: 6 Amino acid sequence of C2 HuVK 1 humanized light chain variable region
(CDR3)
SEQ ID NO: 7 Amino acid sequence of Aβ epitope region 2
SEQ ID NO: 8 Amino acid sequence of Aβ epitope region 1
SEQ ID NO: 9 Amino acid sequence of Aβ epitope region 2 modified
SEQ ID NO: 10 Amino acid sequence of Aβ epitope region 1 modified
SEQ ID NO: 11 Amino acid sequence of Epitope region modified complete
SEQ ID NO: 12 Amino acid sequence of C2 HuVK 1 humanized light chain variable region
SEY ID NO: 13 Amino acid sequence of C2 humanized light chain
SEQ ID NO: 14 Amino acid sequence of humanized C2 light chain constant region
SEQ ID NO: 15 Amino acid sequence of C2 HuVH AF 4 humanized heavy chain variable region
SEQ ID NO: 16 Amino acid sequence of C2 humanized heavy chain
SEQ ID NO: 17: Amino acid sequence of IG GAMMA-4 CHAIN C REGION - modified
SEQ ID NO: 18: Nucleotide sequence of CDR2 of C2 HuVH AF 4 humanised heavy chain variable
region
SEQ ID NO: 19: Nucleotide sequence of CDR3 of C2 HuVH AF 4 humanised heavy chain variable
region
SEQ ID NO: 20: Nucleotide sequence of CDR1 of C2 HuVK 1 humanised light chain variable
region
SEQ ID NO: 21: Nucleotide sequence of C2 HuVK 1 humanized light chain variable region
SEQ ID NO: 22: Nucleotide sequence of C2 humanized light chain
SEQ ID NO: 23: Nucleotide sequence of C2 humanized light chain constant region
SEQ ID NO: 24: Nucleotide sequence of C2 HuVH AF 4 humanized heavy chain variable
region
SEQ ID NO: 25: Nucleotide sequence of C2 humanized heavy chain
SEQ ID NO: 26: Nucleotide sequence of C2 humanized heavy chain constant region
SEQ ID NO: 27: Amino acid sequence of Mouse C2 Light Chain Variable Region
SEQ ID NO: 28: Amino acid sequence of Mouse C2 Heavy Chain Variable Region
SEQ ID NO: 29: Nucleotide sequence of Mouse C2 Light Chain Variable Region
SEQ ID NO: 30: Nucleotide sequence of Mouse C2 Light Chain
SEQ ID NO: 31: Nucleotide sequence of Mouse C2 Heavy Chain Variable Region
SEQ ID NO: 32: Nucleotide sequence of Mouse C2 Heavy Chain
DEFINITIONS
[0219] The terms "polypeptide", "peptide", and "protein", as used herein, are interchangeable
and are defined to mean a biomolecule composed of amino acids linked by a peptide
bond.
[0220] The language "diseases and disorders which are caused by or associated with amyloid
or amyloid-like proteins" includes, but is not limited to, diseases and disorders
caused by the presence or activity of amyloid-like proteins in monomeric, fibril,
or polymeric state, or any combination of the three. Such diseases and disorders include,
but are not limited to, amyloidosis, endocrine tumors, and macular degeneration.
[0221] The term "amyloidosis" refers to a group of diseases and disorders associated with
amyloid plaque formation including, but not limited to, secondary amyloidosis and
age-related amyloidosis such as diseases including, but not limited to, neurological
disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized
by a loss of cognitive memory capacity such as, for example, mild cognitive impairment
(MCI), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis
(Dutch type); the Guam Parkinson-Dementia complex; as well as other diseases which
are based on or associated with amyloid-like proteins such as progressive supranuclear
palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related
dementia, ALS (amyotropic lateral sclerosis), inclusion-body myositis (IBM), Adult
Onset Diabetes, and senile cardiac amyloidosis; and various eye diseases including
macular degeneration, drusen-related optic neuropathy, and cataract due to beta-amyloid
deposition.
[0222] The terms "detecting" or "detected" as used herein mean using known techniques for
detection of biologic molecules such as immunochemical or histological methods and
refer to qualitatively or quantitatively determining the presence or concentration
of the biomolecule under investigation.
[0223] "Polymeric soluble amyloid" refers to multiple aggregated monomers of amyloid peptides,
or of amyloid-like peptides, or of modified or truncated amyloid peptides or of other
derivates of amyloid peptides forming oligomeric or polymeric structures which are
soluble in the mammalian or human body more particularly in the brain, but particularly
to multiple aggregated monomers of amyloid
β (A
β) or of modified or truncated amyloid
β (A
β) peptides or of derivatives thereof, which are soluble in the mammalian or human
body more particularly in the brain.
[0224] "Amyloid
β, A
β or
β-amyloid" is an art recognized term and refers to amyloid
β proteins and peptides, amyloid
β precursor protein (APP), as well as modifications, fragments and any functional equivalents
thereof. In particular, by amyloid
β as used herein is meant any fragment produced by proteolytic cleavage of APP but
especially those fragments which are involved in or associated with the amyloid pathologies
including, but not limited to, A
β1-38, A
β1-39, A
β1-40, A
β1-41 A
β1-42 and A
β1-43.
[0225] The structure and sequences of the amyloid
β peptides as mentioned above are well known to those skilled in the art and methods
of producing said peptides or of extracting them from brain and other tissues are
described, for example, in
Glenner and Wong, Biochem Biophys Res Comm129, 885-890 (1984). Moreover, amyloid
β peptides are also commercially available in various forms.
[0226] By "isolated" is meant a biological molecule free from at least some of the components
with which it naturally occurs.
[0227] The terms "antibody" or "antibodies" as used herein are art-recognized terms and
are understood to refer to molecules or active fragments of molecules that bind to
known antigens, particularly to immunoglobulin molecules and to immunologically active
portions of immunoglobulin molecules, i.e molecules that contain a binding site that
specifically binds an antigen. An immunoglobulin is a protein comprising one or more
polypeptides substantially encoded by the immunoglobulin kappa and lambda, alpha,
gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin
variable region genes. Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define
the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Also subclasses
of the heavy chain are known. For example, IgG heavy chains in humans can be any of
IgG1, IgG2, IgG3 and IgG4 subclass. The immunoglobulin according to the invention
can be of any class (IgG, IgM, IgD, IgE, IgA and IgY) or subclass (IgGl, IgG2, IgG3,
IgG4, IgAl and IgA2) of immunoglobulin molecule.
[0228] As used herein "specifically binds" in reference to an antibody means that the antibody
binds to its target antigen with greater affinity that it does to a structurally different
antigen(s).
[0229] A typical immunoglobulin structural unit is known to comprise a tetramer. Each tetramer
is composed of two identical pairs of polypeptide chains, each pair having one "light"
(about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain
defines a variable region of about 100 to 110 or more amino acids primarily responsible
for antigen recognition. The terms variable light chain (V
L) and variable heavy chain (V
H) refer to these light and heavy chains respectively.
[0230] Antibodies exist as full length intact antibodies or as a number of well-characterized
fragments produced by digestion with various peptidases or chemicals. Thus, for example,
pepsin digests an antibody below the disulfide linkages in the hinge region to produce
F(ab')
2, a dimer of Fab which itself is a light chain joined to V
H-CH
1 by a disulfide bond. The F(ab')
2 may be reduced under mild conditions to break the disulfide linkage in the hinge
region thereby converting the F(ab')
2 dimer into an Fab' monomer. The Fab' monomer is essentially a Fab fragment with part
of the hinge region (see,
Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody
fragments are defined in terms of the digestion of an intact antibody, one of skill
will appreciate that any of a variety of antibody fragments may be synthesized de
novo either chemically or by utilizing recombinant DNA methodology. Thus, the term
antibody, as used herein also includes antibody fragments either produced by the modification
of whole antibodies or synthesized de novo or antibodies and fragments obtained by
using recombinant DNA methodologies.
[0231] "Antibodies" are intended within the scope of the present invention to include monoclonal
antibodies, polyclonal antibodies, chimeric, single chain, bispecific, simianized,
human and humanized antibodies as well as active fragments thereof. Examples of active
fragments of molecules that bind to known antigens include separated light and heavy
chains, Fab, Fab/c, Fv, Fab', and F(ab')
2 fragments, including the products of an Fab immunoglobulin expression library and
epitope-binding fragments of any of the antibodies and fragments mentioned above.
[0232] These active fragments can be derived from an antibody of the present invention by
a number of techniques. For example, monoclonal antibodies can be cleaved with an
enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction
containing Fab fragments can then be collected and concentrated by membrane filtration
and the like. For further description of general techniques for the isolation of active
fragments of antibodies, see for example,
Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982);
Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.
[0233] Recombinantly made antibodies may be conventional full length antibodies, active
antibody fragments known from proteolytic digestion, unique active antibody fragments
such as Fv or single chain Fv (scFv), domain deleted antibodies, and the like. An
Fv antibody is about 50 Kd in size and comprises the variable regions of the light
and heavy chain. A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL
heterodimer which may be expressed from a nucleic acid including VH- and VL-encoding
sequences either joined directly or joined by a peptide-encoding linker. See
Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883. A number of structures for converting the naturally aggregated, but chemically separated
light and heavy polypeptide chains from an antibody V region into an scFv molecule
which will fold into a three dimensional structure substantially similar to the structure
of an antigen-binding site. See, e.g.
U.S. Patent Nos. 5,091,513,
5,132,405 and
4,956,778.
[0234] The combining site refers to the part of an antibody molecule that participates in
antigen binding. The antigen binding site is formed by amino acid residues of the
N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. The antibody
variable regions comprise three highly divergent stretches referred to as "hypervariable
regions" or "complementarity determining regions" (CDRs) which are interposed between
more conserved flanking stretches known as "framework regions" (FRs). In an antibody
molecule, the three hypervariable regions of a light chain (LCDR1, LCDR2, and LCDR3)
and the three hypervariable regions of a heavy chain (HCDR1, HCDR2 and HCDR3) are
disposed relative to each other in three dimensional space to form an antigen binding
surface or pocket. The antibody combining site therefore represents the amino acids
that make up the CDRs of an antibody and any framework residues that make up the binding
site pocket.
[0235] The identity of the amino acid residues in a particular antibody that make up the
combining site can be determined using methods well known in the art. For example,
antibody CDRs may be identified as the hypervariable regions originally defined by
Kabat et al. (see, "
Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department
of Health and Human Services;
Johnson, G and Wu, TT (2001) Kabat Database and its applications: future directions.
Nucleic Acids Research, 29: 205-206; http://immuno.bme.nwa.edu). The positions of the CDRs may also be identified as the structural loop structures
originally described by Chothia and others, (see
Chothia and Lesk, J. Mol. Biol. 196, 901 (1987),
Chothia et al., Nature 342, 877 (1989), and
Tramontano et al., J. Mol. Biol. 215, 175 (1990)). Other methods include the "AbM definition" which is a compromise between Kabat
and Chothia and is derived using Oxford Molecular's AbM antibody modeling software
(now Accelrys) or the "contact definition" of CDRs by
Macallum et al., ("Antibody-antigen interactions: contact analysis and binding site
topography," J Mol Biol. 1996 Oct 11;262(5):732-45). The following chart identifies CDRs based upon various known definitions.
Loop |
Kabat |
AbM |
Chothia |
Contact |
L1 |
L24 -- L34 |
L24 -- L34 |
L24 -- L34 |
L30 -- L36 |
L2 |
L50 -- L56 |
L50 -- L56 |
L50 -- L56 |
L46 -- L55 |
L3 |
L89 -- L97 |
L89 -- L97 |
L89 -- L97 |
L89 -- L96 |
H1 |
H31 -- H35B |
H26 -- H35B |
H26 -- H32..34 |
H30 -- H35B |
(Kabat Numbering) |
H1 |
H31 -- H35 |
H26 -- H35 |
H26 -- H32 |
H30 -- H35 |
(Chothia Numbering) |
H2 |
H50 -- H65 |
H50 -- H58 |
H52 -- H56 |
H47 -- H58 |
H3 |
H95 -- H102 |
H95 -- H102 |
H95 -- H102 |
H93 -- H101 |
General guidelines by which one may identify the CDRs in an antibody from sequence
alone are as follows:
LCDR1:
Start - Approximately residue 24.
Residue before is always a Cys.
Residue after is always a Trp. Typically TRP is followed with TYR-GLN, but also may
be followed by LEU-GLN, PHE-GLN, or TYR-LEU.
Length is 10 to 17 residues.
LCDR2:
Start - 16 residues after the end of L1.
Sequence before is generally ILE-TYR, but also may be VAL-TYR, ILE-LYS, or ILE-PHE.
Length is generally 7 residues.
LCDR3:
Start - generally 33 residues after end of L2.
Residue before is a Cys.
Sequence after is PHE-GLY-X-GLY.
Length is 7 to 11 residues.
HCDR1:
Start - at approximately residue 26 (four residues after a CYS) [Chothia / AbM definition]
Kabat definition starts 5 residues later.
Sequence before is CYS-X-X-X.
Residues after is a TRP, typically followed by VAL, but also followed by ILE, or ALA.
Length is 10 to 12 residues under AbM definition while Chothia definition excludes
the last 4 residues.
HCDR2:
Start - 15 residues after the end of Kabat /AbM definition of CDR-H1.
Sequence before typically LEU-GLU-TRP-ILE-GLY, but a number of variations are possible.
Sequence after is LYS/ARG-LEU/ILE/VAL/PHE/THR/ALA-THR/SER/ILE/ALA
Length is 16 to 19 residues under Kabat definition (AbM definition ends 7 residues
earlier).
HCDR3:
Start -33 residues after end of CDR-H2 (two residues after a CYS).
Sequence before is CYS-X-X (typically CYS-ALA-ARG).
Sequence after is TRP-GLY-X-GLY.
Length is 3 to 25 residues.
[0236] The identity of the amino acid residues in a particular antibody that are outside
the CDRs, but nonetheless make up part of the combining site by having a side chain
that is part of the lining of the combining site (i.e., it is available to linkage
through the combining site), can be determined using methods well known in the art
such as molecular modeling and X-ray crystallography. See e.g.,
Riechmann et al., (1988) Nature, 332:;323-327.
[0237] Chimeric antibodies are those in which one or more regions of the antibody are from
one species of animal and one or more regions of the antibody are from a different
species of animal. A preferred chimeric antibody is one which includes regions from
a primate immunoglobulin. A chimeric antibody for human clinical use is typically
understood to have variable regions from a non-human animal, e.g. a rodent, with the
constant regions from a human. In contrast, a humanized antibody uses CDRs from the
non-human antibody with most or all of the variable framework regions from and all
the constant regions from a human immunoglobulin. A human chimeric antibody is typically
understood to have the variable regions from a rodent. A typical human chimeric antibody
has human heavy constant regions and human light chain constant regions with the variable
regions of both the heavy and light coming from a rodent antibody. A chimeric antibody
may include some changes to a native amino acid sequence of the human constant regions
and the native rodent variable region sequence. Chimeric and humanized antibodies
may be prepared by methods well known in the art including CDR grafting approaches
(see, e.g.,
U.S. Patent Nos. 5,843,708;
6,180,370;
5,693,762;
5,585,089;
5,530,101), chain shuffling strategies (see e.g.,
U.S. Patent No. 5,565,332;
Rader et al., Proc. Natl. Acad. Sci. USA (1998) 95:8910-8915), molecular modeling strategies (
U.S. Patent No. 5,639,641), and the like.
[0238] A "humanized antibody" as used herein in the case of a two chain antibody is one
where at least one chain is humanized. A humanized antibody chain has a variable region
where one or more of the framework regions are human. A humanized antibody which is
a single chain is one where the chain has a variable region where one or more of the
framework regions are human. The non-human portions of the variable region of the
humanized antibody chain or fragment thereof is derived from a non-human source, particularly
a non-human antibody, typically of rodent origin. The non-human contribution to the
humanized antibody is typically provided in form at least one CDR region which is
interspersed among framework regions derived from one (or more) human immunoglobulin(s).
In addition, framework support residues may be altered to preserve binding affinity.
[0239] The humanized antibody may further comprise constant regions (e.g., at least one
constant region or portion thereof, in the case of a light chain, and preferably three
constant regions in the case of a heavy chain). The constant regions of a humanized
antibody if present generally are human.
[0241] A "humanized antibody" may also be obtained by a novel genetic engineering approach
that enables production of affinity-matured human-like polyclonal antibodies in large
animals such as, for example, rabbits and mice. See, e.g.
U.S. Pat No. 6,632,976.
The term constant region (CR) as used herein refers to constant regions genes of the
immunoglobulin. The constant region genes encode the portion of the antibody molecule
which confers effector functions. For Chimeric human antibodies and humanized antibodies,
typically non-human (e.g., murine), constant regions are substituted by human constant
regions. The constant regions of the subject chimeric or humanized antibodies are
typically derived from human immunoglobulins. The heavy chain constant region can
be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further,
heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are
responsible for different effector functions and thus, by choosing the desired heavy
chain constant region, antibodies with desired effector function can be produced.
Constant regions that may be used within the scope of this invention are gamma 1 (IgG1),
particularly an Fc region of the gamma 1 (IgG1) isotype, gamma 3 (IgG3) and especially
gamma 4 (IgG4). The light chain constant region can be of the kappa or lambda type,
preferably of the kappa type. In one embodiment the light chain constant region is
the human kappa constant chain (
Heiter et al. (1980) Cell 22:197-207) and the heavy constant chain is the human IgG4 constant chain.
[0242] The term "monoclonal antibody" is also well recognized in the art and refers to an
antibody that is the product of a single cloned antibody producing cell. Monoclonal
antibodies are typically made by fusing a normally short-lived, antibody-producing
B cell to a fast-growing cell, such as a cancer cell (sometimes referred to as an
"immortal" cell). The resulting hybrid cell, or hybridoma, multiplies rapidly, creating
a clone that produces the antibody.
[0243] For the purpose of the present invention, "monoclonal antibody" is also to be understood
to comprise antibodies that are produced by a mother clone which has not yet reached
full monoclonality.
[0244] "Functionally equivalent antibody" is understood to refer to an antibody which substantially
shares at least one major functional property with an antibody mentioned above and
herein described comprising: binding specificity to the
β-amyloid protein, particularly to the A
β1-42 protein, and more particularly to the 16-21 epitope region of the A
β1-42 protein, immunoreactivity
in vitro, inhibition of aggregation of the A
β1-42 monomers into high molecular polymeric fibrils and/or disaggregation of preformed
A
β1-42 polymeric fibrils, and/or a
β-sheet breaking property and alleviating the effects of amyloidosis, a group of diseases
and disorders associated with amyloid plaque formation including secondary amyloidosis
and age-related amyloidosis such as diseases including, but not limited to, neurological
disorders such as Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome, hereditary
cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex;
as well as other diseases which are based on or associated with amyloid-like proteins
such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,
Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult
Onset Diabetes; senile cardiac amyloidosis; endocrine tumors, and others, including
macular degeneration, when administered prophylactically or therapeutically. The antibodies
can be of any class such as IgG, IgM, or IgA, etc or any subclass such as IgG1, IgG2a,
etc and other subclasses mentioned herein above or known in the art, but particularly
of the IgG4 class. Further, the antibodies can be produced by any method, such as
phage display, or produced in any organism or cell line, including bacteria, insect,
mammal or other type of cell or cell line which produces antibodies with desired characteristics,
such as humanized antibodies. The antibodies can also be formed by combining a Fab
portion and an Fc region from different species.
[0245] The term "hybridize" as used refers to conventional hybridization conditions, preferably
to hybridization conditions at which 5xSSPE, 1% SDS, 1xDenhardts solution is used
as a solution and/or hybridization temperatures are between 35°C and 70°C, preferably
65°C. After hybridization, washing is preferably carried out first with 2xSSC, 1%
SDS and subsequently with 0.2xSSC at temperatures between 35°C and 70°C, preferably
at 65°C (regarding the definition of SSPE, SSC and Denhardts solution see Sambrook
et al. loc. cit.). Stringent hybridization conditions as for instance described in
Sambrook et al, supra, are particularly preferred. Particularly preferred stringent
hybridization conditions are for instance present if hybridization and washing occur
at 65°C as indicated above. Non-stringent hybridization conditions, for instance with
hybridization and washing carried out at 45°C are less preferred and at 35°C even
less.
[0246] "Homology" between two sequences is determined by sequence identity. If two sequences
which are to be compared with each other differ in length, sequence identity preferably
relates to the percentage of the nucleotide residues of the shorter sequence which
are identical with the nucleotide residues of the longer sequence. Sequence identity
can be determined conventionally with the use of computer programs such as the Bestfit
program (
Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive Madison, WI 53711). Bestfit utilizes the local homology algorithm of
Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, in order to find the segment having the highest sequence identity between two sequences.
When using Bestfit or another sequence alignment program to determine whether a particular
sequence has for instance 95% identity with a reference sequence of the present invention,
the parameters are preferably so adjusted that the percentage of identity is calculated
over the entire length of the reference sequence and that homology gaps of up to 5%
of the total number of the nucleotides in the reference sequence are permitted. When
using Bestfit, the so-called optional parameters are preferably left at their preset
("default") values. The deviations appearing in the comparison between a given sequence
and the above-described sequences of the invention may be caused for instance by addition,
deletion, substitution, insertion or recombination. Such a sequence comparison can
preferably also be carried out with the program "fasta20u66" (version 2.0u66, September
1998 by William R. Pearson and the University of Virginia; see also
W.R. Pearson (1990), Methods in Enzymology 183, 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the "default"
parameter settings maybe used.
[0247] The antibody according to the invention may be an immunoglobulin or antibody, which
is understood to have each of its binding sites identical (if multivalent) or, in
the alternative, may be a "bispecific" or "bifunctional antibody".
[0249] The term "fragment" refers to a part or portion of an antibody or antibody chain
comprising fewer amino acid residues than an intact or complete antibody or antibody
chain. Fragments can be obtained
via chemical or enzymatic treatment of an intact or complete antibody or antibody chain.
Fragments can also be obtained by recombinant means. Exemplary fragments include Fab,
Fab', F(ab')2, Fabc and/or Fv fragments. The term "antigen-binding fragment" refers
to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes
with intact antibody (
i.e., with the intact antibody from which they were derived) for antigen binding (
i.e., specific binding).
[0250] Binding fragments are produced by recombinant DNA techniques, or by enzymatic or
chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab',
F(ab')
2, Fabc, Fv, single chains, and single-chain antibodies.
[0251] "Fragment" also refers to a peptide or polypeptide comprising an amino acid sequence
of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues,
at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues,
at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues,
at least 50 contiguous amino acid residues, at least 60 contiguous amino residues,
at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues,
at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues,
at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues,
at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues,
or at least contiguous 250 amino acid residues of the amino acid sequence of another
polypeptide. In a specific embodiment, a fragment of a polypeptide retains at least
one function of the polypeptide.
[0252] The term "antigen" refers to an entity or fragment thereof which can bind to an antibody.
An immunogen refers to an antigen which can elicit an immune response in an organism,
particularly an animal, more particularly a mammal including a human. The term antigen
includes regions known as antigenic determinants or epitopes which refers to a portion
of the antigen (which are contacted or which play a significant role in supporting
a contact reside in the antigen responsible for antigenicity or antigenic determinants.
[0253] As used herein, the term "soluble" means partially or completely dissolved in an
aqueous solution.
[0254] Also as used herein, the term "immunogenic" refers to substances which elicit the
production of antibodies, T-cells and other reactive immune cells directed against
an antigen of the immunogen.
[0255] An immune response occurs when an individual produces sufficient antibodies, T-cells
and other reactive immune cells against administered immunogenic compositions of the
present invention to moderate or alleviate the disorder to be treated.
[0256] The term immunogenicity as used herein refers to a measure of the ability of an antigen
to elicit an immune response (humoral or cellular) when administered to a recipient.
The present invention is concerned with approaches that reduce the immunogenicity
of the subject human chimeric or humanized antibodies.
[0257] Humanized antibody of reduced immunogenicity refers to a humanized antibody exhibiting
reduced immunogenicity relative to the parent antibody, e.g., the murine antibody.
[0258] Humanized antibody substantially retaining the binding properties of the parent antibody
refers to a humanized antibody which retains the ability to specifically bind the
antigen recognized by the parent antibody used to produce such humanized antibody.
Preferably the humanized antibody will exhibit the same or substantially the same
antigen-binding affinity and avidity as the parent antibody. Ideally, the affinity
of the antibody will not be less than 10% of the parent antibody affinity, more preferably
not less than about 30%, and most preferably the affinity will not be less than 50%
of the parent antibody. Methods for assaying antigen-binding affinity are well known
in the art and include half-maximal binding assays, competition assays, and Scatchard
analysis. Suitable antigen binding assays are described in this application.
[0259] A "back mutation" is a mutation introduced in a nucleotide sequence which encodes
a humanized antibody, the mutation results in an amino acid corresponding to an amino
acid in the parent antibody (
e.g., donor antibody, for example, a murine antibody). Certain framework residues from
the parent antibody may be retained during the humanization of the antibodies of the
invention in order to substantially retain the binding properties of the parent antibody,
while at the same time minimizing the potential immunogenicity of the resultant antibody.
In one embodiment of the invention, the parent antibody is of mouse origin. For example,
the back mutation changes a human framework residue to a parent murine residue. Examples
of framework residues that may be back mutated include, but are not limited to, canonical
residues, interface packing residues, unusual parent residues which are close to the
binding site, residues in the "Vernier Zone" (which forms a platform on which the
CDRs rest) (
Foote & Winter, 1992, J. Mol. Biol. 224, 487-499), and those close to CDR H3.
[0260] As used herein a "conservative change" refers to alterations that are substantially
conformationally or antigenically neutral, producing minimal changes in the tertiary
structure of the mutant polypeptides, or producing minimal changes in the antigenic
determinants of the mutant polypeptides, respectively, as compared to the native protein.
When referring to the antibodies and antibody fragments of the invention, a conservative
change means an amino acid substitution that does not render the antibody incapable
of binding to the subject receptor. Those of ordinary skill in the art will be able
to predict which amino acid substitutions can be made while maintaining a high probability
of being conformationally and antigenically neutral. Such guidance is provided, for
example in
Berzofsky, (1985) Science 229:932-940 and
Bowie et al. (1990) Science 247:1306-1310. Factors to be considered that affect the probability of maintaining conformational
and antigenic neutrality include, but are not limited to: (a) substitution of hydrophobic
amino acids is less likely to affect antigenicity because hydrophobic residues are
more likely to be located in a protein's interior; (b) substitution of physiochemically
similar, amino acids is less likely to affect conformation because the substituted
amino acid structurally mimics the native amino acid; and (c) alteration of evolutionarily
conserved sequences is likely to adversely affect conformation as such conservation
suggests that the amino acid sequences may have functional importance. One of ordinary
skill in the art will be able to assess alterations in protein conformation using
well-known assays, such as, but not limited to microcomplement fixation methods (
Wasserman et al. (1961) J. Immunol. 87:290-295;
Levine et al. (1967) Meth. Enzymol. 11:928-936) and through binding studies using conformation-dependent monoclonal antibodies (
Lewis et al. (1983) Biochem. 22:948-954).
[0261] Further, the term "therapeutically effective amount" refers to the amount of antibody
which, when administered to a human or animal, which is sufficient to result in a
therapeutic effect in said human or animal. The effective amount is readily determined
by one of skill in the art following routine procedures.
[0262] As used herein, the terms "treat," "prevent," "preventing," and "prevention" refer
to the prevention of the recurrence or onset of one or more symptoms of a disorder
in a subject resulting from the administration of a prophylactic or therapeutic agent.
Construction of Humanized Antibodies
[0263] The present invention provides novel uses and compositions comprising highly specific
and highly effective antibodies having the ability to specifically recognize and bind
to specific epitopes from a range of
β-amyloid antigens. The antibodies enabled by the teaching of the present invention
are particularly useful for the treatment of amyloidosis, a group of diseases and
disorders associated with amyloid plaque formation including secondary amyloidosis
and age-related amyloidosis including, but not limited to, neurological disorders
such as Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome, hereditary
cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex;
as well as other diseases which are based on or associated with amyloid-like proteins
such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,
hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, HIV-related
dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac
amyloidosis; endocrine tumors, and others, including macular degeneration, to name
just a few.
[0264] A fully humanized or reshaped variable region according to the present invention
may be created within the scope of the invention by first designing a variable region
amino acid sequence that contains CDRs derived from murine antibody ACI-01-Ab7C2 (named
"mC2" throughout the application and deposited 01 December 2005 with the "Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in Braunschweig, Mascheroder
Weg 1 B, 38124 Branuschweig, under the provisions of the Budapest Treaty and given
accession no DSM ACC2750) embedded in human-derived framework sequences. The non-human-,
particularly the rodent-derived CDRs, which may be obtained from the antibody according
to the present invention, provide the desired specificity. Accordingly, these residues
are to be included in the design of the reshaped variable region essentially unchanged.
Any modifications should thus be restricted to a minimum and closely watched for changes
in the specificity and affinity of the antibody. On the other hand, framework residues
in theory can be derived from any human variable region.
[0265] In order to create a reshaped antibody which shows an acceptable or an even improved
affinity, a human framework sequences should be chosen, which is equally suitable
for creating a reshaped variable region and for retaining antibody affinity.
[0266] In order to achieve this goal, the best-fit strategy was developed. As it is known
that the framework sequences serve to hold the CDRs in their correct spatial orientation
for interaction with antigen, and that framework residues can sometimes even participate
in antigen binding, this strategy aims at minimizing changes that may negatively effect
the three-dimensional structure of the antibody by deriving the human framework sequence
used for antibody reshaping from the human variable region that is most homologous
or similar to the non-human-, particularly the rodent-derived variable region. This
will also maximise the likelihood that affinity will be retained in the reshaped antibody.
[0267] At its simplest level, the "best fit" strategy involves comparing the donor rodent
V-region with all known human V-region amino acid sequences, and then selecting the
most homologous to provide the acceptor framework regions for the humanization exercises.
In reality there are several other factors which should be considered, and which may
influence the final selection of acceptor framework regions. Molecular modelling predictions
may be used in this regard prior to any experimental work in an attempt to maximise
the affinity of the resultant reshaped antibody. Essentially, the goal of the modelling
is to predict which key residues (if any) of the most homologous human framework should
be left as in the rodent to obtain the best affinity in the reshaped antibody.
[0268] In one embodiment of the invention, the CDRs are obtainable from mouse monoclonal
antibody, particularly from mouse monoclonal antibody ACI-01-Ab7C2 (named "mC2" throughout
the application) described in co-pending application
EP 05 02 7092.5 filed 12.12.2005.
[0269] Hybridoma cells FP-12H3-C2, producing mouse monoclonal antibody ACI-01-Ab7C2 (named
"mC2" and hC2 for the humanized C2 antibody, throughout the application) were deposited
01 December 2005 in co-pending application no
EP05027092.5 with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in Braunschweig,
Mascheroder Weg 1 B, 38124 Braunschweig, under the provisions of the Budapest Treaty
and given accession no DSM ACC2750.
[0270] The mouse antibody may be raised against a supramolecular antigenic construct comprising
an antigenic peptide corresponding to the amino acid sequence of the
β-amyloid peptide, particularly of
β-amyloid peptide A
β1-15, A
β1-16 and A
β1-16(Δ14), modified with a hydrophobic moiety such as, for example, palmitic acid or a hydrophilic
moiety such as, for example, polyethylene glycol (PEG) or a combination of both, wherein
the hydrophobic and hydrophilic moiety, respectively, is covalently bound to each
of the termini of the antigenic peptide through at least one, particularly one or
two amino acids such as, for example, lysine, glutamic acid and cysteine or any other
suitable amino acid or amino acid analogue capable of serving as a connecting device
for coupling the hydrophobic and hydrophilic moiety to the peptide fragment. When
a PEG is used as the hydrophilic moiety, the free PEG termini is covalently bound
to phosphatidylethanolamine or any other compound suitable to function as the anchoring
element, for example, to embed the antigenic construct in the bilayer of a liposome.
[0271] In particular, a mouse antibody may be raised against a supramolecular antigenic
construct comprising an antigenic peptide corresponding to the amino acid sequence
of the
β-amyloid peptide A
β1-16 modified with a hydrophilic moiety such as, for example, polyethylene glycol (PEG)
hydrophilic moiety is covalently bound to each of the termini of the antigenic peptide
through at least one, particularly one or two amino acids such as, for example, lysine,
glutamic acid and cysteine or any other suitable amino acid or amino acid analogue
capable of serving as a connecting device for coupling the hydrophobic and hydrophilic
moiety to the peptide fragment. When a PEG is used as the hydrophilic moiety, the
free PEG termini are covalently bound to phosphatidylethanolamine or any other compound
suitable to function as the anchoring element, for example, to embed the antigenic
construct in the bilayer of a liposome.
[0272] In an embodiment of the invention, a humanized antibody or a fragment thereof is
provided which comprises in the variable region at least one CDR of non-human origin
embedded in one or more human- or primate-derived framework regions and combined with
a constant region derived from a human or primate source antibody, which chimeric
antibody or a fragment thereof, or a humanized antibody or a fragment thereof is capable
of specifically recognizing and binding
β-amyloid monomeric peptide.
[0274] In the process for preparing a humanized antibody according to the invention, the
amino acid sequences of the C2 heavy chain and light chain variable regions (V
H and V
K) are compared to rodent antibody V
H and V
K sequences in the NCBI and Kabat databases.
[0275] The closest match mouse germ line gene to C2 V
K is bbl, Locus MMU231201, (Schable
et al, 1999). A comparison reveals that two amino acids differ from this germ line sequence,
both located within CDRL1. Mature murine antibodies with similar, but not identical,
sequence can be found. Several have an identical CDRL2 and identical CDRL3, but the
CDRL1 of C2 seems to be unique. Comparison with human germ line V
K sequences shows that genes from subgroup V
KII are the best match for C2 V
K (Cox
et al, 1994). C2 V
K can thus be assigned to Kabat subgroup MuV
KII.Sequence.
[0276] DPK15 together with the human J region HuJ
K1 may be selected to provide the acceptor framework sequences for the humanized V
K.
[0277] The residues at the interface between the variable light and heavy chains have been
defined (
Chothia et al, 1985 J. Mol. Biol., 186, 651-663). These are usually retained in the reshaped antibody. The Phe at position 87 of
mouse C2 V
K is unusual at the interface, where a Tyr is more common in the V
KII subgroup, indicating that this framework residue may be important for antibody
activity. Tyr 87 is present in the human germline and humanized C2VK.
[0278] The humanized V
K sequences thus may be designed such that the C2HuVK1 consists of mouse C2 V
K CDRs with frameworks from DPK 15 and human J
K1. In a specific embodiment of the invention, murine residues may be substituted in
the human framework region at positions 45, and/or 87. In the CDR2 region obtainable
from a mouse monoclonal antibody, particularly murine antibody ACI-01-Ab7C2, amino
acid substitutions may be made at Kabat positions 50 and/or 53. Residue 45 may be
involved in supporting the conformation of the CDRs. Residue 87 is located at the
interface of the V
H and V
K domains. Therefore these residues may be critical for maintenance of antibody binding.
[0279] The closest match mouse germ line gene to C2 V
H AF is VH7183, Locus AF120466, (Langdon
et al, 2000). Comparison with human germ line V
H sequences shows that genes from subgroup V
HIII are the best match for C2 V
H. C2 V
H AF can be assigned to Kabat subgroup MuV
HIIID. Sequence DP54 together with the human J region HuJ
H6 can be selected to provide the acceptor framework sequences for the humanized V
H.
The comparison shows that there are nine amino acid differences between the C2 VH
sequences and the human acceptor germ line sequence DP54 and J
H6, most being located within CDRH2. Mature murine antibodies with identical or similar
(one residue different) CDRH1 or with similar CDRH2 (one residue different) are found,
but none with all three CDRs identical to C2 V
H AF. CDRH3 of C2 antibody is unusually short, consisting of only three residues. However,
other antibodies are found in the database with CDRH3 of this length. Residue 47 of
C2 V
H is Leu rather than the more common Trp, and residue 94 is Ser rather than the normal
Arg, indicating that these framework residues may be important for antibody activity.
[0280] Various humanized V
H sequences may be designed. C2HuVH1 consists of C2 V
H AF CDRs with frameworks from DP54 and HuJ
H6. In a specific embodiment of the invention, murine residues may be substituted in
the human framework region at positions 47 or 94 or both. Residue 47 in framework
2 makes contact both with the CDRs and with the V
K domain. Residue 94 may be involved in supporting the conformation of the CDRs. Therefore
these residues may be critical for maintenance of antibody binding.
[0281] Different HCVR and LCVR regions may be designed which comprise the non-human CDRs
obtainable from the donor antibody, for example, a murine antibody, embedded into
the native or modified human- or primate-derived framework regions. The modification
may particularly concern an exchange of one or more amino acid residues within the
framework region by non-human residues, particularly murine residues, more commonly
found in this position in the respective subgroups or by residues which have similar
properties to the ones more commonly found in this position in the respective subgroups.
[0282] The modification of the framework region the framework sequences serve to hold the
CDRs in their correct spatial orientation for interaction with antigen, and that framework
residues can sometimes even participate in antigen binding. In one embodiment of the
invention measures are taken to further adapt the selected human framework sequences
to make them most similar to the sequences of the rodent frameworks in order to maximise
the likelihood that affinity will be retained in the reshaped antibody.
[0283] Accordingly, murine residues in the human framework region may be substituted. In
particular, murine residues may be substituted in the human framework region of the
Heavy Chain Variable (HCVR) region at positions 47 or 94 or both and in the human
framework region of the Light Chain Variable (LCVR) region at positions 45 and/or
87. In the CDR2 region obtainable from a mouse monoclonal antibody, particularly murine
antibody ACI-01-Ab7C2, amino acid substitutions may be made at Kabat positions 50
and/or 53..
[0284] The residues found in the above indicated positions in the human framework region
may be exchanged by murine residues more commonly found in this position in the respective
subgroups. In particular, the Trp in Kabat position 47 in the human- or primate-derived
framework region of the Heavy Chain Variable Region as shown in SEQ ID NO: 15 may
be replaced by an Leu or by an amino acid residue that has similar properties and
the substitution of which leads to alterations that are substantially conformationally
or antigenically neutral, producing minimal changes in the tertiary structure of the
mutant polypeptides, or producing minimal changes in the antigenic determinants. In
particular, the Trp in Kabat position 47 in the human- or primate-derived framework
region of the Heavy Chain Variable Region as shown in SEQ ID NO: 15 may further be
replaced by an amino acid selected from the group consisting of norleucine, Ile, Val,
Met, Ala, and Phe, particularly by Ile. Alternative conservative substitutions may
be contemplated which are conformationally and antigenically neutral.
The Arg in Kabat position 94 in the human- or primate-derived framework region of
the Heavy Chain Variable Region as shown in SEQ ID NO: 15 may be replaced by Ser or
by an amino acid residue that has similar properties and the substitution of which
leads to alterations that are substantially conformationally or antigenically neutral,
producing minimal changes in the tertiary structure of the mutant polypeptides, or
producing minimal changes in the antigenic determinants. In particular, the Arg in
Kabat position 94 in the human- or primate-derived framework region of the Heavy Chain
Variable Region as shown in SEQ ID NO: 15 may alternatively be replaced by Thr.
[0285] In another alternative, both residues may be replaced in the humanized antibody.
The Gln in Kabat position 45 in the human- or primate-derived framework region of
the Light Chain Variable Region as shown in SEQ ID NO: 12 may be replaced by Lys or
by an amino acid residue that has similar properties and the substitution of which
leads to alterations that are substantially conformationally or antigenically neutral,
producing minimal changes in the tertiary structure of the mutant polypeptides, or
producing minimal changes in the antigenic determinants. In particular, the Gln in
Kabat position 45 in the human- or primate-derived framework region of the Light Chain
Variable Region as shown in SEQ ID NO: 12 may be replaced by an amino acid selected
from the group consisting of Arg, Gln, and Asn, particularly by Arg.
[0286] The Leu in Kabat position 50 in the human- or primate-derived framework region of
the Light Chain Variable Region as shown in SEQ ID NO: 12 may be replaced by Lys or
by an amino acid residue that has similar properties and the substitution of which
leads to alterations that are substantially conformationally or antigenically neutral,
producing minimal changes in the tertiary structure of the mutant polypeptides, or
producing minimal changes in the antigenic determinants. In particular, the Leu in
Kabat position 50 in the human- or primate-derived framework region of the Light Chain
Variable Region as shown in SEQ ID NO: 12 may be replaced by an amino acid selected
from the group consisting of Arg, Gln, and Asn, particularly by Arg.
[0287] The Asn in Kabat position 53 in the human- or primate-derived framework region of
the Light Chain Variable Region as shown in SEQ ID NO: 12 may be replaced by His and
Gln or by an amino acid residue that has similar properties and the substitution of
which leads to alterations that are substantially conformationally or antigenically
neutral, producing minimal changes in the tertiary structure of the mutant polypeptides,
or producing minimal changes in the antigenic determinants. In particular, the Asn
in Kabat position 53 in the human- or primate-derived framework region of the Light
Chain Variable Region as shown in SEQ ID NO: 12 may be replaced by an amino acid selected
from the group consisting of Gln, His, Lys and Arg.
[0288] The Thr in Kabat position 87 in the human- or primate-derived framework region of
the Light Chain Variable Region as shown in SEQ ID NO: 12 may be replaced by Phe or
by an amino acid residue that has similar properties and the substitution of which
leads to alterations that are substantially conformationally or antigenically neutral,
producing minimal changes in the tertiary structure of the mutant polypeptides, or
producing minimal changes in the antigenic determinants. In particular, the Tyr in
Kabat position 87 in the human- or primate-derived framework region of the Light Chain
Variable Region as shown in SEQ ID NO: 12 may be replaced by an amino acid selected
from the group consisting of Leu, Val, Ile, and Ala, particularly by Leu.
[0289] The so obtained variable region comprising at least one CDR of non-human origin embedded
in one or more human- or primate-derived framework regions may then be combined with
a constant region derived from a human or primate source antibody, particularly with
human IgG4 or κ constant regions respectively. The IgG4 constant region may be modified
by, for example, changing Serine at position 228 in the hinge region to Proline (HuIgG4
Ser-Pro). This mutation stabilizes the interchain disulphide bond and prevents the
formation of half molecules that may occur in native human IgG4 preparations. The
IgG4 constant region may be further modified by deletion of the terminal Lys in position
439 as shown in SEQ ID NO: 16.
[0290] The modified variable regions may be constructed by method known in the art such
as, for example overlapping PCR recombination. The expression cassettes for the chimeric
antibody, C2 ChV
H AF and C2 ChV
K, may be used as templates for mutagenesis of the framework regions to the required
sequences. Sets of mutagenic primer pairs are synthesized encompassing the regions
to be altered. The humanized V
H and V
K expression cassettes produced may be cloned into appropriate cloning vectors know
in the art such as, for example, pUC19. After the entire DNA sequence is confirmed
to be correct for each V
H and V
K, the modified heavy and light chain V-region genes can be excised from the cloning
vector as expression cassettes. These can then be transferred to appropriate expression
vectors such as pSV
gpt and pSV
hyg which include human IgG4 Ser-Pro or κ constant regions respectively.
EXPRESSION VECTORS
[0291] Expression vector
pSVgpt is based on pSV
2gpt (Mulligan and Berg, 1980) and includes the ampicillin resistance gene for selection
in bacterial cells, the
gpt gene for selection in mammalian cells, the murine heavy chain immunoglobulin enhancer
region, genomic sequence encoding the constant region gene and SV40 poly A sequences.
The heavy chain variable region for expression is inserted as a
HindIII to
BamHI fragment.
[0292] Expression vector pSVhyg includes the ampicillin resistance gene for selection in
bacterial cells, the
hyg gene for selection in mammalian cells, the murine heavy chain immunoglobulin enhancer
region, genomic sequence encoding the kappa constant region gene and including the
kappa enhancer and SV40 poly A sequences. The light chain variable region for expression
is inserted as a
HindIII to
BamHI fragment.
[0293] The DNA sequence is then to be confirmed to be correct for the humanized V
H and V
K in the expression vectors.
[0294] For antibody production the humanized heavy and light chain expression vectors may
be introduced into appropriate production cell lines know in the art such as, for
example, NS0 cells. Introduction of the expression vectors may be accomplished by
co-transfection via electroporation or any other suitable transformation technology
available in the art. Antibody producing cell lines can then be selected and expanded
and humanized antibodies purified. The purified antibodies can then be analyzed by
standard techniques such as SDS-PAGE.
ANTIBODY WITH IMPROVED AFFINITY, SPECIFICITY, STABILITY
[0295] The CDRL2 sequence ("KVSNRFS") of the mouse C2 antibody may be modified slightly
without adversely affecting antibody activity. Conservative substitutions may be made
through exchange of R for K at position 50 and S for N at position 53. The two alternative
CDRL2 sequences are therefore "RVSNRFS" and "KVSSRFS", respectively. These are incorporated
into the murine V
K sequence with no other changes, as C2 VK-R and C2 VK-S, respectively.
[0296] The affinity, specificity and stability of an antibody according to the invention
as described herein before or a fragment thereof can be modified by change of its
glycosylation profile or pattern resulting in improved therapeutic values.
[0297] To achieve this change in glycosylation pattern, host cells may be engineered such
that they are capable of expressing a preferred range of a glycoprotein-modifying
glycosyl transferase activity which increases complex N-linked oligosaccharides carrying
bisecting GIcNAc. Further, modified glycoforms of glycoproteins may be obtained, for
example antibodies, including whole antibody molecules, antibody fragments, or fusion
proteins that include a region equivalent to the Fc region of an immunoglobulin, having
an enhanced Fc-mediated cellular cytotoxicity.
PHARMACEUTICAL PREPARATION AND ADMINISTRATION
[0299] The antibodies according to the invention, but particularly a monoclonal antibody
according the invention, can be prepared in a physiologically acceptable formulation
and may comprise a pharmaceutically acceptable carrier, diluent and/or excipient using
known techniques. For example, the antibody according to the invention and as described
herein before in particular, the monoclonal antibody including any functional parts
thereof is combined with a pharmaceutically acceptable carrier, diluent and/or excipient
to form a therapeutic composition. Suitable pharmaceutical carriers, diluents and/or
excipients are well known in the art and include, for example, phosphate buffered
saline solutions, water, emulsions such as oil/water emulsions, various types of wetting
agents, sterile solutions, etc.
[0300] Formulation of the pharmaceutical composition according to the invention can be accomplished
according to standard methodology know to those skilled in the art.
[0301] The compositions of the present invention may be administered to a subject in the
form of a solid, liquid or aerosol at a suitable, pharmaceutically effective dose.
Examples of solid compositions include pills, creams, and implantable dosage units.
Pills may be administered orally. Therapeutic creams may be administered topically.
Implantable dosage units may be administered locally, for example, at a tumor site,
or may be implanted for systematic release of the therapeutic composition, for example,
subcutaneously. Examples of liquid compositions include formulations adapted for injection
intramuscularly, subcutaneously, intravenously, intra-arterially, and formulations
for topical and intraocular administration. Examples of aerosol formulations include
inhaler formulations for administration to the lungs.
[0302] The compositions may be administered by standard routes of administration. In general,
the composition may be administered by topical, oral, rectal, nasal, interdermal,
intraperitoneal, or parenteral (for example, intravenous, subcutaneous, or intramuscular)
routes. In addition, the composition may be incorporated into sustained release matrices
such as biodegradable polymers, the polymers being implanted in the vicinity of where
delivery is desired, for example, at the site of a tumor. The method includes administration
of a single dose, administration of repeated doses at predetermined time intervals,
and sustained administration for a predetermined period of time.
[0303] A sustained release matrix, as used herein, is a matrix made of materials, usually
polymers which are degradable by enzymatic or acid/base hydrolysis or by dissolution.
Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
The sustained release matrix desirably is chosen by biocompatible materials such as
liposomes, polylactides (polylactide acid), polyglycolide (polymer of glycolic acid),
polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides,
poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic
acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids,
amino acids such phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene,
polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of
one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers
of lactic acid and glycolic acid).
[0304] It is well know to those skilled in the pertinent art that the dosage of the composition
will depend on various factors such as, for example, the condition of being treated,
the particular composition used, and other clinical factors such as weight, size,
sex and general health condition of the patient, body surface area, the particular
compound or composition to be administered, other drugs being administered concurrently,
and the route of administration.
[0305] The composition may be administered in combination with other compositions comprising
an biologically active substance or compound, particularly at least one compound selected
from the group consisting of compounds against oxidative stress, anti-apoptotic compounds,
metal chelators, inhibitors of DNA repair such as pirenzepin and metabolites, 3-amino-1-propanesulfonic
acid (3APS), 1,3-propanedisulfonate (1,3PDS), α-secretase activators,
β- and
γ -secretase inhibitors, tau proteins, neurotransmitter,
β-sheet breakers, attractants for amyloid beta clearing / depleting cellular components,
inhibitors of N-terminal truncated amyloid beta including pyroglutamated amyloid beta
3-42, anti-inflammatory molecules, "atypical antipsychotics" such as, for example
clozapine, ziprasidone, risperidone, aripiprazole or olanzapine or cholinesterase
inhibitors (ChEIs) such as tacrine, rivastigmine, donepezil, and/or galantamine, M1
agonists and other drugs including any amyloid or tau modifying drug and nutritive
supplements such as, for example, vitamin B12, cysteine, a precursor of acetylcholine,
lecithin, choline, Ginkgo biloba, acyetyl-L-carnitine, idebenone, propentofylline,
or a xanthine derivative, together with an antibody according to the present invention
and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an
excipient and procedures for the treatment of diseases.
[0306] Proteinaceous pharmaceutically active matter may be present in amounts between 1
ng and 10 mg per dose. Generally, the regime of administration should be in the range
of between 0.1
µg and 10 mg of the antibody according to the invention, particularly in a range 1.0
µg to 1.0 mg, and more particularly in a range of between 1.0
µg and 100
µg, with all individual numbers falling within these ranges also being part of the
invention. If the administration occurs through continuous infusion a more proper
dosage may be in the range of between 0.01
µg and 10 mg units per kilogram of body weight per hour with all individual numbers
falling within these ranges also being part of the invention.
[0307] Administration will generally be parenterally, eg intravenously. Preparations for
parenteral administration include sterile aqueous or non-aqueous solutions, suspensions
and emulsions. Non-aqueous solvents include without being limited to it, propylene
glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous solvents may be chosen from the group consisting
of water, alcohol/aqueous solutions, emulsions or suspensions including saline and
buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles
include fluid and nutrient replenishers, electrolyte replenishers (such as those based
on Ringer's dextrose) and others. Preservatives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, inert gases, etc.
[0308] The pharmaceutical composition may further comprise proteinaceous carriers such as,
for example, serum albumin or immunoglobulin, particularly of human origin. Further
biologically active agents may be present in the pharmaceutical composition of the
invention dependent on its the intended use.
[0309] When the binding target is located in the brain, certain embodiments of the invention
provide for the antibody or active fragment thereof to traverse the blood-brain barrier.
Certain neurodegenerative diseases are associated with an increase in permeability
of the blood-brain barrier, such that the antibody or active fragment thereof can
be readily introduced to the brain. When the blood-brain barrier remains intact, several
art-known approaches exist for transporting molecules across it, including, but not
limited to, physical methods, lipid-based methods, and receptor and channel-based
methods.
[0310] Physical methods of transporting the antibody or active fragment thereof across the
blood-brain barrier include, but are not limited to, circumventing the blood-brain
barrier entirely, or by creating openings in the blood-brain barrier. Circumvention
methods include, but are not limited to, direct injection into the brain (see, e.g.,
Papanastassiou et al., Gene Therapy 9: 398-406 (2002)) and implanting a delivery device in the brain (see, e.g.,
Gill et al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, Guildford Pharmaceutical). Methods of creating openings in
the barrier include, but are not limited to, ultrasound (see, e.g.,
U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., by administration of hypertonic mannitol (
Neuwelt, E. A., Implication of the Blood-Brain Barrier and its Manipulation, Vols
1 & 2, Plenum Press, N.Y. (1989))), permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g.,
U.S. Patent Nos. 5,112,596,
5,268,164,
5,506,206, and
5,686,416), and transfection of neurons that straddle the blood-brain barrier with vectors
containing genes encoding the antibody or antigen-binding fragment (see, e.g.,
U.S. Patent Publication No. 2003/0083299).
[0311] Lipid-based methods of transporting the antibody or active fragment thereof across
the blood-brain barrier include, but are not limited to, encapsulating the antibody
or active fragment thereof in liposomes that are coupled to antibody binding fragments
that bind to receptors on the vascular endothelium of the blood-brain barrier (see,
e.g.,
U.S. Patent Application Publication No. 20020025313), and coating the antibody or active fragment thereof in low-density lipoprotein
particles (see, e.g., U.S. Patent Application Publication No.
20040204354) or apolipoprotein E (see, e.g., U.S. Patent Application Publication No.
20040131692).
[0312] Receptor and channel-based methods of transporting the antibody or active fragment
thereof across the blood-brain barrier include, but are not limited to, using glucocorticoid
blockers to increase permeability of the blood-brain barrier (see, e.g.,
U.S. Patent Application Publication Nos. 2002/0065259,
2003/0162695, and
2005/0124533); activating potassium channels (see, e.g.,
U.S. Patent Application Publication No. 2005/0089473), inhibiting ABC drug transporters (see, e.g.,
U.S. Patent Application Publication No. 2003/0073713); coating antibodies with a transferrin and modulating activity of the one or more
transferrin receptors (see, e.g.,
U.S. Patent Application Publication No. 2003/0129186), and cationizing the antibodies (see, e.g.,
U.S. Patent No. 5,004,697).
DETECTION/DIAGNOSIS
[0313] In a further embodiment the present invention provides methods and kits for the detection
and diagnosis of amyloid-associated diseases or conditions. These methods include
known immunological methods commonly used for detecting or quantifying substances
in biological samples or in an
in situ condition.
[0314] Diagnosis of an amyloid-associated disease or condition in a patient may be achieved
by detecting the immunospecific binding of a monoclonal antibody or an active fragment
thereof to an epitope of the amyloid protein in a sample or
in situ, which includes bringing the sample or a specific body part or body area suspected
to contain the amyloid protein into contact with an antibody which binds an epitope
of the amyloid protein, allowing the antibody to bind to the amyloid protein to form
an immunological complex, detecting the formation of the immunological complex and
correlating the presence or absence of the immunological complex with the presence
or absence of amyloid protein in the sample or specific body part or area.
[0315] Biological samples that may be used in the diagnosis of an amyloid-associated disease
or condition are, for example, fluids such as serum, plasma, saliva, gastric secretions,
mucus, cerebrospinal fluid, lymphatic fluid and the like or tissue or cell samples
obtained from an organism such as neural, brain, cardiac or vascular tissue. For determining
the presence or absence of the amyloid protein in a sample any immunoassay known to
those of ordinary skill in the art. (See
Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New
York 1988 555-612) may be used such as, for example, assays which utilize indirect detection methods
using secondary reagents for detection, ELISA's and immunoprecipitation and agglutination
assays. A detailed description of these assays is, for example, given in
WO96/13590 to Maertens and Stuyver, Zrein et al. (1998) and
WO96/29605.
[0316] For
in situ diagnosis, the antibody or any active and functional part thereof may be administered
to the organism to be diagnosed by methods known in the art such as, for example,
intravenous, intranasal, intraperitoneal, intracerebral, intraarterial injection such
that a specific binding between the antibody according to the invention with an eptitopic
region on the amyloid protein may occur. The antibody/antigen complex may be detected
through a label attached to the antibody or a functional fragment thereof.
[0317] The immunoassays used in diagnostic applications typically rely on labelled antigens,
antibodies, or secondary reagents for detection. These proteins or reagents can be
labelled with compounds generally known to those skilled in the art including enzymes,
radioisotopes, and fluorescent, luminescent and chromogenic substances including colored
particles, such as colloidal gold and latex beads. Of these, radioactive labelling
can be used for almost all types of assays and with most variations. Enzyme-conjugated
labels are particularly useful when radioactivity must be avoided or when quick results
are needed. Fluorochromes, although requiring expensive equipment for their use, provide
a very sensitive method of detection. Antibodies useful in these assays include monoclonal
antibodies, polyclonal antibodies, and affinity purified polyclonal antibodies.
Alternatively, the antibody may be labelled indirectly by reaction with labelled substances
that have an affinity for immunoglobulin, such as protein A or G or second antibodies.
The antibody may be conjugated with a second substance and detected with a labelled
third substance having an affinity for the second substance conjugated to the antibody.
For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate
detected using labelled avidin or streptavidin. Similarly, the antibody may be conjugated
to a hapten and the antibody-hapten conjugate detected using labelled anti-hapten
antibody.
[0318] Those of ordinary skill in the art will know of these and other suitable labels which
may be employed in accordance with the present invention. The binding of these labels
to antibodies or fragments thereof can be accomplished using standard techniques commonly
known to those of ordinary skill in the art. Typical techniques are described by
Kennedy, J. H., et al.,1976 (Clin. Chim. Acta 70:1-31), and
Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 81:1-40). Coupling techniques mentioned in the latter are the glutaraldehyde method, the
periodate method, the dimaleimide method, and others.
[0319] Current immunoassays utilize a double antibody method for detecting the presence
of an analyte, wherein. The antibody is labeled indirectly by reactivity with a second
antibody that has been labeled with a detectable label. The second antibody is preferably
one that binds to antibodies of the animal from which the monoclonal antibody is derived.
In other words, if the monoclonal antibody is a mouse antibody, then the labeled,
second antibody is an anti-mouse antibody. For the monoclonal antibody to be used
in the assay described below, this label is preferably an antibody-coated bead, particularly
a magnetic bead. For the polyclonal antibody to be employed in the immunoassay described
herein, the label is preferably a detectable molecule such as a radioactive, fluorescent
or an electrochemiluminescent substance.
[0320] An alternative double antibody system often referred to as fast format systems because
they are adapted to rapid determinations of the presence of an analyte, may also be
employed within the scope of the present invention. The system requires high affinity
between the antibody and the analyte. According to one embodiment of the present invention,
the presence of the amyloid protein is determined using a pair of antibodies, each
specific for amyloid protein. One of said pairs of antibodies is referred to herein
as a "detector antibody" and the other of said pair of antibodies is referred to herein
as a "capture antibody". The monoclonal antibody of the present invention can be used
as either a capture antibody or a detector antibody. The monoclonal antibody of the
present invention can also be used as both capture and detector antibody, together
in a single assay. One embodiment of the present invention thus uses the double antibody
sandwich method for detecting amyloid protein in a sample of biological fluid. In
this method, the analyte (amyloid protein) is sandwiched between the detector antibody
and the capture antibody, the capture antibody being irreversibly immobilized onto
a solid support. The detector antibody would contain a detectable label, in order
to identify the presence of the antibody-analyte sandwich and thus the presence of
the analyte.
[0321] Exemplary solid phase substances include, but are not limited to, microtiter plates,
test tubes of polystyrene, magnetic, plastic or glass beads and slides which are well
known in the field of radioimmunoassay and enzyme immunoassay. Methods for coupling
antibodies to solid phases are also well known to those skilled in the art. More recently,
a number of porous material such as nylon, nitrocellulose, cellulose acetate, glass
fibers and other porous polymers have been employed as solid supports.
[0322] The present invention also relates to a diagnostic kit for detecting amyloid protein
in a biological sample comprising a composition as defined above. Moreover, the present
invention relates to the latter diagnostic kit which, in addition to a composition
as defined above, also comprises a detection reagent as defined above. The term "diagnostic
kit" refers in general to any diagnostic kit known in the art. More specifically,
the latter term refers to a diagnostic kit as described in Zrein et al. (1998).
[0323] It is still another object of the present invention to provide novel immunoprobes
and test kits for detection and diagnosis of amyloid-associated diseases and conditions
comprising antibodies according to the present invention. For immunoprobes, the antibodies
are directly or indirectly attached to a suitable reporter molecule, e.g., an enzyme
or a radionuclide. The test kit includes a container holding one or more antibodies
according to the present invention and instructions for using the antibodies for the
purpose of binding to amyloid protein to form an immunological complex and detecting
the formation of the immunological complex such that presence or absence of the immunological
complex correlates with presence or absence of amyloid protein.
EXAMPLES
Materials
[0324] The development and preparation of mouse monoclonal antibody ACI-01-Ab7C2 (named
"mC2" and hC2 for the humanized C2 antibody, throughout the application) is described
in co-pending application
EP 05 02 7092.5 filed 12.12.2005.
[0325] Hybridoma cells FP-12H3-C2, producing mouse monoclonal antibody ACI-01-Ab7C2 (named
"mC2" and hC2 for the humanized C2 antibody, throughout the application) were deposited
01 December 2005 in co-pending application no
EP05027092.5 with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in Braunschweig,
Mascheroder Weg 1 B, 38124 Braunschweig, under the provisions of the Budapest Treaty
and given accession no DSM ACC2750.
[0326] Hybridoma cells were cultured in Dulbecco's modified Eagle Medium (DMEM) supplemented
with 10% foetal bovine serum and antibiotics (Penicillin/Streptomycin). The isotype
of the antibody produced was checked and found to be mouse IgG2b/kappa, as expected.
Assay
[0327] An ELISA for binding to Amyloid Beta provided a reliable measure of the potency of
C2 antibodies. Positive control antibodies, murine FP-12H3-C2 antibody (Genovac Lot
No: AK379/01), and standard Chemicon antibody 1560 (Lot no: 0508008791).
Choice of human constant regions
[0328] As immune system recruitment is not desirable for the clinical antibody candidate,
the selected human constant region for the heavy chain was human IgG4, modified to
change Serine at position 228 in the hinge region to Proline (HuIgG4 Ser-Pro). This
mutation stabilizes the interchain disulphide bond and prevents the formation of half
molecules that may occur in native human IgG4 preparations. The antibody expressed
from the production cell lines will also have the terminal lysine removed. The sequences
of human constant regions HuIgG4 Ser-Pro and human Kappa are given in SEQ ID NO: 17
and 14, respectively.
Sequence Listing
[0329]
<110> AC Immune S.A.
<110> Genentech, Inc.
<120> Humanized Antibody
<130> M1967 EP/A/1 BS
<140> EP 07840408.4
<141> 2007-07-13
<150> EP 06014730.3
<151> 2006-07-14
<150> EP 06020765.1
<151> 2006-10-02
<150> US 60/943,289
<151> 2007-06-11
<150> US 60/943,499
<151> 2007-06-12
<160> 32
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> Mus musculus
<220>
<223> C2 HuVH AF 4 humanised heavy chain variable region (CDR1)
<400> 1
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0001)
<210> 2
<211> 17
<212> PRT
<213> Mus musculus
<220>
<223> C2 HuVH AF 4 humanised heavy chain variable region (CDR2)
<400> 2
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0002)
<210> 3
<211> 3
<212> PRT
<213> Mus musculus
<220>
<223> C2 HuVH AF 4 humanised heavy chain variable region (CDR3)
<400> 3
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0003)
<210> 4
<211> 16
<212> PRT
<213> Mus musculus
<220>
<223> C2 HuVK 1 humanised light chain variable region (CDR1)
<400> 4
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0004)
<210> 5
<211> 7
<212> PRT
<213> Mus musculus
<220>
<223> C2 HuVK 1 humanised light chain variable region (CDR2)
<400> 5
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0005)
<210> 6
<211> 9
<212> PRT
<213> Mus musculus
<220>
<223> C2 HuVK 1 humanised light chain variable region (CDR3)
<400> 6
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0006)
<210> 7
<211> 6
<212> PRT
<213> Homo sapiens
<220>
<223> A-betta epitope, region 2
<400> 7
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0007)
<210> 8
<211> 5
<212> PRT
<213> Homo sapiens
<220>
<223> A-beta epitope, region 1
<400> 8
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0008)
<210> 9
<211> 6
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa can be Ala, Val, Leu, norleucine, Met, Phe, or Ile
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa can be Ala, Val, Leu, Ser, or Ile
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa can be Glu or Asp
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa can be Glu or Asp
<400> 9
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0009)
<210> 10
<211> 5
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa can be His, Asn, Gln, Lys, or Arg
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa can be Asn or Gln
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa can be Ala, Val, Leu, norleucine, Met, Phe, or Ile
<400> 10
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0010)
<210> 11
<211> 10
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa can be His, Asn, Gln, Lys, or Arg
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa can be Asn or Gln
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa can be Ala, Val, Leu, norleucine, Met, Phe, or Ile
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa can be Ala, Val, Leu, Ser, or Ile
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa can be Glu or Asp
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa can be Glu or Asp
<400> 11
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0011)
<210> 12
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> artificial humanized C2 HuVK 1 variable light chain
<400> 12
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0012)
<210> 13
<211> 219
<212> PRT
<213> artificial sequence
<220>
<223> artificial humanized C2 light chain
<400> 13
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0013)
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0014)
<210> 14
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> artificial humanized C2 light chain constant region
<400> 14
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0015)
<210> 15
<211> 112
<212> PRT
<213> artificial sequence
<220>
<223> artificial humanized C2 HuVH AF 4 variable heavy chain
<400> 15
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0016)
<210> 16
<211> 439
<212> PRT
<213> artificial sequence
<220>
<223> artificial humanized C2 heavy chain
<400> 16
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0017)
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0018)
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0019)
<210> 17
<211> 326
<212> PRT
<213> Homo sapiens
<220>
<223> IG GAMMA-4 CHAIN C REGION - modified
<400> 17
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0020)
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0021)
<210> 18
<211> 51
<212> DNA
<213> Mus musculus
<220>
<223> C2 HuVH AF 4 humanised heavy chain variable region (CDR2)
<400> 18
agcatcaata gtaatggtgg tagcacctat tatccagaca gtgtgaaggg c 51
<210> 19
<211> 9
<212> DNA
<213> Mus musculus
<220>
<223> C2 HuVH AF 4 humanised heavy chain variable region (CDR3)
<400> 19
ggtgactac 9
<210> 20
<211> 49
<212> DNA
<213> Mus musculus
<220>
<223> C2 HuVK 1 humanised light chain variable region (CDR1)
<400> 20
agatctagtc agagccttgt atatagtaat ggagacacct atttacatt 49
<210> 21
<211> 336
<212> DNA
<213> artificial sequence
<220>
<223> artificial humanized C2 Hu VK 1 variable light chain
<400> 21
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0022)
<210> 22
<211> 657
<212> DNA
<213> artificial sequence
<220>
<223> artificial humanized C2 light chain
<400> 22
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0023)
<210> 23
<211> 321
<212> DNA
<213> Homo sapiens
<220>
<223> artificial humanized C2 light chain constant region
<400> 23
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0024)
<210> 24
<211> 336
<212> DNA
<213> artificial sequence
<220>
<223> artificial humanized C2 HuVH AF variable heavy chain
<400> 24
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0025)
<210> 25
<211> 1317
<212> DNA
<213> artificial sequence
<220>
<223> artificial humanized C2 heavy chain
<400> 25
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0026)
<210> 26
<211> 981
<212> DNA
<213> Homo sapiens
<220>
<223> artificial humanized C2 heavy chain constant region
<400> 26
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0027)
<210> 27
<211> 112
<212> PRT
<213> Mus musculus
<400> 27
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0028)
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0029)
<210> 28
<211> 112
<212> PRT
<213> Mus musculus
<400> 28
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0030)
<210> 29
<211> 336
<212> DNA
<213> Mus musculus
<400> 29
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0031)
<210> 30
<211> 417
<212> DNA
<213> Mus musculus
<400> 30
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0032)
<210> 31
<211> 336
<212> DNA
<213> Mus musculus
<400> 31
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0033)
<210> 32
<211> 408
<212> DNA
<213> Mus musculus
<400> 32
![](https://data.epo.org/publication-server/image?imagePath=2017/51/DOC/EPNWB1/EP11192705NWB1/imgb0034)